WO2015094330A1 - Alpha-enolase specific antibodies and methods of uses in cancer therapy - Google Patents

Alpha-enolase specific antibodies and methods of uses in cancer therapy Download PDF

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Publication number
WO2015094330A1
WO2015094330A1 PCT/US2013/076877 US2013076877W WO2015094330A1 WO 2015094330 A1 WO2015094330 A1 WO 2015094330A1 US 2013076877 W US2013076877 W US 2013076877W WO 2015094330 A1 WO2015094330 A1 WO 2015094330A1
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Prior art keywords
antibody
enol
seq
cells
sequence
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PCT/US2013/076877
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French (fr)
Inventor
Shih-Chong Tsai
Neng-Yao Shih
Ta-Tung Yuan
Ko-Jiunn Liu
Shih-Chi TSENG
Chih-Yung Hu
Hsin-Yun WANG
Li-Tzong Chen
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Development Center For Biotechnology
National Health Research Institutes
Dcb-Usa Llc
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Application filed by Development Center For Biotechnology, National Health Research Institutes, Dcb-Usa Llc filed Critical Development Center For Biotechnology
Priority to CN201380082061.0A priority Critical patent/CN106488932B/en
Priority to EP13899506.3A priority patent/EP3083697B1/en
Priority to PCT/US2013/076877 priority patent/WO2015094330A1/en
Priority to RU2016129166A priority patent/RU2656153C1/en
Priority to ES13899506T priority patent/ES2729797T3/en
Priority to AU2013408259A priority patent/AU2013408259B2/en
Priority to CA2949045A priority patent/CA2949045C/en
Priority to TW103144368A priority patent/TWI549967B/en
Publication of WO2015094330A1 publication Critical patent/WO2015094330A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates to methodsforgeneration and use ofantibodiesthat bind specifically to the human alpha-enolase protein (ENOl).
  • ENOl human alpha-enolase protein
  • Tumors result from aberrant, unrestrained proliferation of a single cell, generating a clone of transformed cells.
  • Tumor cells may express unique antigens that can be recognized by the immune system.
  • Tumor-associated antigens includemutated oncogenes, mutated normal cellular proteins, aberrantly expressed cellular proteins, abnormal cell-surface proteins, and oncogenic viral proteins.
  • the immune system views these tumor-associated antigens as non-self andean produce antibodies to eradicate these foreign antigen-bearing tumor cells, while sparing the healthy cells. Therefore, identification of immunogenic tumor-associated antigens may be used as targets for clinical prognostic or therapeutic applications in cancer treatment.
  • Certain malignancies may be identified by pleural effusion, which is excess fluid in the space between the lung and chest wall.
  • Lung carcinoma, breast carcinoma, and lymphoma cause about 75% of all malignant pleural effusions.
  • Malignant pleural effusion may be enriched with lymphocytic infiltrates and tumor cells.
  • Tumor-associated immune complexes or autoantibodies such as anti-p53, antinuclear, and anti-L-Myc antibodies, have been found in effusion fluids and are associated with poor prognosis.
  • lung tumor-associated antigens have also been identified in malignant effusion, including, cytokeratin 19 fragments, neuron-specific enolase (EN02), squamous cell carcinoma antigen, and soluble HLA-I, etc.
  • Alpha-enolase (enolase- 1, ENOl) is a multiple functional protein, which was first found as a key enzyme of the glycolysis pathways. Under normal conditions, ENOl is expressed in the cytosol. However, ENOl is also found to express on the cell surfacesof many cancer cellsas a plasminogen receptor and on activated hematopoietic cells, such as neutrophils, lymphocytes and monocytes. It is known that the up-regulation of plasminogen receptor proteins can induce a cascade response of the urokinase plasminogen activationsystem (uPAS).
  • uPAS urokinase plasminogen activationsystem
  • the urokinase plasminogen activator system consists of the urokinase plasminogen activator (uPA), its cognate receptor (uPAR) and two specific inhibitors, the plasminogen activator inhibitor 1 (PAI-1) and plasminogen activator inhibitor 2 (PAI-2).
  • Urokinase plasminogen activator converts plasminogen proenzyme into an active serine protease, plasmin.
  • Plasmin is involved in a number of tissue remodeling processes, such as basement membrane (BM) and extracellular matrix (ECM) remodeling, which is required in tumor progression and metastasis.
  • BM basement membrane
  • ECM extracellular matrix
  • the uPAS may be involved in the neoplastic evolution, affecting tumor angiogenesis, malignant cell proliferation, adhesion and migration, intra- vascularization, and growth at the metastatic site.
  • ENOl is a potential target for cancer therapy.
  • Embodiments of the invention relate to targeted binding agents that specifically bind human ENOl, thereby inhibiting ligand (e.g., plasminogen) binding to ENOl.
  • ligand e.g., plasminogen
  • targeted binding agents of the invention can inhibit plasminogen activation, leading to reduced degradation of extracellular matrix, which in turn prevents or reduces dissociation of cancer cells from the extracellular matrix. Therefore, targeted binding agents in accordance with embodiments of the invention can be used to inhibit tumor growth and metastasis.
  • Mechanisms by which this can be achieved may include,but are not limited to, inhibition of binding of a ligand (such as plasminogen) to its receptor ENOl, or abrogation of inter-reactions between the receptor ENOl and its ligands, thereby reducing the effective concentration of ENOl.
  • a ligand such as plasminogen
  • a targeted binding agent is anantibody that can bindto humanENOlto prevent its ligands (e.g., plasminogen) from binding to ENOl. Preventing binding of plasminogen to the receptor can preventplasminogenactivation. This results in the inhibition of the urokinase plasminogen activation system (uPAS) in the extracellular matrix of cancer cells.
  • ligands e.g., plasminogen
  • the antibody may bind ENOl with high affinities, such as with a K d of less than 0.3nM.Such tight binding agents can inhibit ENOl with high efficiencies.
  • a targeted binding agent is an antibody that can bind to human ENOl and inhibit induced plasmin activity on cancer cells with high efficiencies, such as 80%, 90%>, or 100% inhibition.
  • the inhibition assays may be performed by inducing ENOl expression (hence, plasminogen activation) in a cancer cell (such asU937 human lymphomacells) by treatmentwith 10 microgram/mL lipopolysaccharide(LPS) for 5 hours. Inhibition of such induced plasmin activity may be assayed with an antibody at a suitable concentration. Using an antibody of the invention, such inhibition may be detectedat antibody concentrations as low as 20 microgram/ml or less.
  • a targeted binding agent is an antibody that can bind to human ENOl .
  • Such an antibody may be used to inhibit the invasion activity of a cancer cell.
  • antibodies of the invention can inhibit greater than 50%, 60%, or 70% ofthe invasion activity of human non-small cell lung carcinoma CLl-5 cells at antibody concentrations as low as 50 microgram/ml or less.
  • a targeted binding agent is an antibody that can bind to humanENOl to inhibit extracellular matrix degradation, thereby inhibiting cancer cell dissociation from the extracellular matrix.
  • an antibody of the invention can inhibit greater than 40%, 50%, or 60% of plasminogen mediated dissociation of CLl-5 cells from collagen or fibronectin at antibody concentrations as low as 50 microgram/ml or less.
  • a targeted binding agent [0013] In accordance with embodiments of the invention, a targeted binding agent
  • CDR complementarity determining region
  • targeted binding agent i.e. an antibody
  • CDR complementarity determining region
  • a targeted binding agent is an antibody, which may be a monoclonal antibody or a polyclonal antibody.
  • an antibodythat can bindhuman ENOlprotein comprises a light chain amino acid sequence comprising any one of LCDR1, LCDR2 or LCDR3 sequence included in sequence 2.
  • an antibodythat can bind human ENOlprotein comprises a light chain amino acid sequence comprising any two of LCDR1, LCDR2 or LCDR3 sequence included in sequence 2(that is,LCDRl and LCDR2, LCDR1 and LCDR3 or LCDR2 and LCDR3).
  • an antibodythat can bind human ENOlprotein comprisesa light chain amino acid sequencesthat comprisesLCDRl, LCDR2 and LCDR3 sequencesincluded in sequence 2.
  • an antibody may be a humanized antibody ora fully human monoclonal antibody.
  • an antibodythat can bind human ENOl protein comprises a heavy chain amino acid sequence comprising any one of HCDRl, HCDR2 or hCDR3 sequence includedin sequence 1.
  • an antibodythat can bind human ENOl protein comprises a heavy chain amino acid sequence that comprises any two of HCDRl, HCDR2 or HCDR3 sequence included in n sequence 1 (that is,HCDRl and HCDR2, HCDRl and HCDR3 or HCDR2 and HCDR3).
  • an antibodythat can bind human ENOl protein comprises a heavy chain amino acid sequencethat comprisesHCDRl , HCDR2 and HCDR3 sequencesincluded insequence 1.
  • an antibody may be a humanized antibody or a fully human monoclonal antibody.
  • an antibody that can bind human ENOl protein comprises a light chain amino acid sequence having a CDR comprising one of the CDR sequences includedin sequence 2.
  • an antibody can bind human ENOl protein and comprises a heavy chain amino acid sequence having a CDR comprising one of the sequences shown in sequences 1.
  • an antibody may be a humanized or a fully human monoclonal antibody.
  • an antibodythat can bind human ENOl protein comprises a heavy chain amino acid sequence having one of the CDR sequences included in sequence 1 and a light chain amino acid sequence having one of the CDR sequences included in sequence 2.
  • an antibody may be a humanized or a fully human monoclonal antibody.
  • a targeted binding agent i.e. an antibody
  • said targeted binding agent comprises a heavy chain amino acid sequence having at least one of the CDR sequences included in sequence 1 and a light chain amino acid sequence having at least one of the CDR sequences included in sequence 2.
  • a targeted binding agent can bind to an epitopecomprising the amino acid sequence 296 FD Q D D W G A W Q F TA 309 (SEQ ID NO:9 ) or 326 R I A K A V N EK S 336 (SEQ ID NO:10 ) of human ENOl protein (GenBank: AAH50642.1).
  • said targeted binding agent comprises a heavy chain amino acid sequence having at least one of the CDR sequences included in sequence 1 and a light chain amino acid sequence having at least one of the CDR sequences included in sequence 2.
  • a binding agent of the invention may comprise an antigen-binding site within a non-antibody molecule.
  • a binding agent maycomprise one or more CDRs e.g. a set of CDRs in a non-antibody protein scaffold, as discussed further below.
  • Some embodiments of the invention relate to methods for assaying the level of human ENOl protein in a patient or a patient sample.
  • a methodof the invention comprises contacting an anti-ENOl antibodywith a biological sample from a patient, and detecting the level of binding between said antibody and human ENOl protein in said sample.
  • the biological sample is blood or plasma.
  • compositions comprising a targeted binding agent, which may include an antibody or a functional fragment thereof, and a pharmaceutically acceptable carrier.
  • Still further embodiments of the invention relate to methods for effectively treating a subject (e.g., human or animal) suffering from an ENOl disease or disorder.
  • the method may include selecting a subject in need of a treatment for a neoplastic or non-neoplastic disease, and administering to the subject a therapeutically effective dose of an antibody (which may be a humanized or a fully human monoclonal antibody) that specifically binds to ENOl protein.
  • an antibody which may be a humanized or a fully human monoclonal antibody
  • the antibody of the invention can be used to treat a human ENOl protein-relateddisease or disorder.
  • a human ENOl protein related disease or disorder may be any condition arising from aberrant activation or expression of human ENOl protein. Examples of such diseases include where human ENOl protein aberrantly interacts with its ligands, thereby altering cell-adhesion or cell signaling properties. This alteration in cell adhesion or cell signaling properties can result in neoplastic diseases or some immune diseases.
  • a human ENOl protein-related disease may be a neoplastic disease, such aslung, breast, pancreas, liver, colorectal, and prostate cancers.
  • a targeted binding agent (e.g., an antibody)can bind to the peptide comprising the amino acid sequence FD Q D D W G A W Q K F T 308 (SEQ ID NO:9) or 326 K R I A K A V N EK S 336 (SEQ ID NO:10) of human ENOl protein (GenBank: AAH50642.1)and can be used to treat a human ENOl protein-related disease or disorder noted above.
  • Additional embodiments of the invention relate to methods for inhibiting
  • ENOl -induced cell dissociationfrom extracellular matrix of cancersin a subject may include selecting a subject (e.g., a human or an animal) in need of treatment for ENOl -induced cell dissociation, and administering to said subject a therapeutically effective dose of an antibody, wherein said antibody specifically binds toENOl.
  • the antibody may be a humanized or fully human monoclonal antibody.
  • Further embodiments of the invention relate to the uses of an antibody in the preparation of a medicament for the treatment of an ENOl -related disease or disorder in a subject (e.g., a human or an animal), wherein said antibody specifically binds toENOl .
  • the antibody may be a humanized or fully human monoclonal antibody.
  • the targeted binding agents described herein can be used for the preparation of a medicament for the treatment of ENOl protein-induced cell dissociationfrom extracellular matrix in an animal, wherein said antibody specifically binds toENOl.
  • the antibody may be a humanized or fully human monoclonal antibody.
  • Some embodiments of the invention described herein relate to monoclonal antibodiesthat bindhuman ENOland affect human ENOl functions.
  • Other embodiments of the invention relate to anti ENOl antibody preparations with desirable properties for therapy applications. Such properties may include a high binding affinity for ENOl, the ability to neutralize ENOl activity in vitro and in vivo, and the ability to inhibit ENOl induced cell dissociation,growth and metastasis of tumors.
  • the invention relates to anantibody that can bindto human ENOlwith very high affinity (i.e., low 3 ⁇ 4).
  • a human, rabbit, mouse, chimeric or humanized antibody that is capable of binding ENOl with a Kjless than about 10 "5 , 10 ⁇ 6 , 10 "7 , 10 ⁇ 8 , 10 "9 , 10 "10 or about 10 " ⁇ ⁇ , or any range or value therebetween.
  • Affinity and/or avidity measurements can be performed using ELISA and/or BIACORE, as described herein or according to techniques known in the art.
  • the anti-ENOl antibody may be a full-length antibody (e.g., having an intact human Fc region) or an antibody fragment (e.g., a Fab, Fab' or F(ab') 2 , FV or Dab (Dabs are the smallest functional binding units of human antibodies).
  • the antibody may be manufactured from a hybridoma that secretes the antibody, or from a recombinantly produced cell that has been transformed or transfected with a gene or genes encoding the antibody.
  • inventions relate to isolated nucleic acid molecules encoding any of the antibodies described herein, vectors having isolated nucleic acid molecules encoding anti-ENOl antibody or a host cell transformed with any of such nucleic acid molecules.
  • some embodiments of the invention relate to a method for producing an anti-ENOl antibody by culturing host cells under conditions wherein a nucleic acid molecule is expressed to produce the antibody, followed by recovering the antibody.
  • embodiments of the invention may also include any nucleic acid molecule which encodes an antibody or fragment of an antibody of the invention including nucleic acid sequences optimized for increasing yields of antibodies or fragments thereof when transfected into host cells for antibody production.
  • Further embodiments of the invention mayrelate to methodsfor producing high affinity antibodies to human ENOl by immunizing a mammal with human ENOl protein, a fragment thereof, and one or more orthologous sequences or fragments thereof.
  • Other embodiments relate to the generation and identification of isolated antibodies that can bind specifically to human ENOl . Inhibition of the biological activity of ENOl can be effected by these antibodies to prevent ENOl induced cell dissociation, invasion and other desired effects of cancers.
  • compositions having an effective amount of an anti-ENOl antibody.
  • the composition may further comprise a pharmaceutically acceptable carrier or diluent.
  • the anti-ENOl antibody, or a fragment thereof is conjugated to a therapeutic agent.
  • the therapeutic agent can be, for example, a toxin or a radioisotope.
  • the methods may include administering to a patient an effective amount of an anti-ENOl antibody.
  • the anti-ENOl antibody can be administered alone, or can be administered in combination with additional antibodies or chemotherapeutic drug or radiation therapy.
  • a monoclonal, oligoclonal or polyclonal mixture of ENOl antibody that block cell dissociation can be administered in combination with a drug shown to inhibit tumor cell proliferation directly.
  • the method can be performed in vivo and the patient is preferably a human patient.
  • the method concerns the treatment of an ENOl -related disease or disorder including, but not limited to, neoplastic diseases, such as lung, breast, pancreas, liver, colorectal, prostate cancers and or solid tumors.
  • Some embodiments of the invention relate to a method for monitoring cancer development.
  • the method may comprise determining the abundance of alpha-enolase proteins (ENOl) in a sample (e.g., cancer cells), wherein an increased level of ENOl correlates with cancer severity.
  • the abundance may be determined by measuring binding of an ENOl specific antibody to the ENOl proteins.
  • Some embodiments of the invention relate to a method for detecting cancer.
  • Such a method may comprise determining the abundance of ENOl -specific antibodies in serum samples, wherein a low level of ENOl -specific antibodies indicates the presence of a malignant tumor.
  • FIGs. 1A and IB show results from studies ofcell surface location of ENOl in malignant cells using flowcytometry (FIG. 1A) and immunostaining (FIG. IB), respectively. Detailed procedures were performed as described in Example l . The data show that ENOl is located on cell surface in malignant cells (NHRI-L89 & CA926), but not in normal lung epithelial cells (NHBE & SAEC).
  • FIG. 2 shows the inhibition of invasion activity of highly-invasive CLl-5 lung adenocarcinoma cells by polyclonal antibody against cell-surface ENOl. Detailed procedures were performed as described in Example 2. The data show that administrating anti-ENOl polyclonal antibody attenuates the invasion of CLl-5.
  • FIG. 3 shows that apolyclonal antibody against human ENOl attenuates tissue invasion capability of CL1-5F4 cells in lung.
  • the administration of antibody in an animal and the lung metastatic colony formation was monitored 12 and 19 days after tumor injection using an IVIS system.
  • the detailed procedures were performed as described in Example 3.
  • the data show that ENOl polyclonal antibody inhibits the migration of CL1-5F4 to lung in vivo.
  • FIG. 4 shows the generation of anti-ENOl antibody hybridomas andverification of each monoclonal antibody clones by flow cytometry.
  • the proceduresforthe immunization of mice and generation of hybridoma, and the production of each antibody and verification of antibody by flow cytometry were described in Example 4.
  • the data show that all5 hybridoma antibodies recognize the surface ENOl on theCLl-5F4 lung adenocarcinoma cells.
  • FIG. 5A shows ENOl binding in ELISAof 5 antibodies isolated from acites of individual hybridomas. Ammonium sulfate purification, protein A column purification, and SDS-PAGE purification were performed as described in Example 5. The data in FIG. 5B show theK d values of 5 different anti human ENOl antibodies.
  • FIG. 6 shows the results of U937 fibrinolytic assay of 5 antibodies isolated from acites of individual hybridomas, respectively.
  • the induction of ENOl expression by LPS in human U937 lymphoma cell line and the plasminactivity assay were performed as described in Example 6. These data also show that 5 different anti-ENOl antibodies have different inhibition activities againstplasminogen receptor of ENOl andthat the inhibition activitiescorrelate withthe 3 ⁇ 4 values.
  • FIG. 7 shows results of inhibition of invasion activities of CLl-5 cells treated with 5 different antibodies isolated from acites of individual hybridomas, respectively. The detailedprocedureswere described in Example 7. These data show that all 5 different anti-ENOl antibodies can inhibit the invasion activity of CLl-5 cells. Interestingly, even though clone 8 has aiQabout 7.2-foldshigher than that of clone 10, the inhibition of invasionactivity against CLl-5 cells byclone 8 is similarto that of clone 10.
  • FIG. 8 A shows results of invasion activities of CLl-5 cells treated with different concentrations of ENlOmAb isolated from hybridoma. Detailedprocedures were performed as described in Example 8. These data show thatENlOmAb antibody inhibits the invasion activity of CLl-5 in a dose-dependent manner.
  • FIG. 8B shows results of invasion activities of U937 cells treated with different concentrations of EN10 mAb isolated from hybridoma,after the surface ENOl expression of cells was induced by LPS. Detailedprocedures were performed as described in Example 8. These data show that the EN10 mAb inhibits the invasion activity of U937 cells in a dose-dependent manner.
  • FIG. 9 shows that EN10 mAb recognizes the cell surface ENOl on U937 cells treated with LPS. Detailed procedures were performed as described in Example 9.
  • FIG. 10A shows the adhesion activity of CLl-5 lung carcinoma cells to matrix proteins.
  • the adhesion assay was performed as described in Example 10. These data show that CLl-5 cells have higher adhesion activities to collagen and fibronectin.
  • FIG. 10B shows results of inhibition of CLl-5 cell dissociationfromfibronectin treated with the EN10 mAb.
  • the cell associated adhesion assay was performed as described in Example 10. These data show that the EN10 mAb inhibits the cell dissociation activity of CLl-5 from fibronectinin a dose-dependent manner.
  • FIG. IOC shows results of inhibition of CLl-5 cell dissociationfromcollagen treated with the EN10 mAb.
  • the cell associated adhesion assay was performed as described in Example 10. These data show that the EN10 mAbinhibits the cell dissociation activity of CLl-5 fromcollagen ina dose-dependent manner.
  • FIG. 11A depicts the variable heavy chain region amino acid sequence of
  • EN10 mAb (SEQ ID NO: 1).
  • the framework regions (FR1, FR2, FR3, and FR4) and CDRs (HCDR1, HCDR2, and HCDR3) are indicated. Cloning the EN10 mAb was performed as described in Example 11.
  • FIG. 1 IB depicts the variable light chain region amino acid sequence of EN10 mAb(SEQ ID NO: 2).
  • the framework regions (FR1, FR2, FR3, and FR4) and CDRs (LCDR1, LCDR2, and LCDR3) are indicated. Cloning the EN10 mAb was performed as described in Example 11.
  • FIG. 12A shows the EN10 mAbbinding activities of deletion mutants of
  • ENOl The binding epitope of EN 10 mAb is locatedbetween the amino residue number 293 and 434 of human ENOl protein.
  • the large portion deletion of ENOl to determine the binding region of EN10 mAb was performed as described in Example 12.
  • FIG. 12B shows the 12% SDS PAGE of 6 C-terminal deletion mutant protein of ENOl purified from E.coli. The detailedproceduresfor thepurification of ENOl deletion mutants aredescribed in Example 12.
  • FIG. 12C shows the EN 10 mAbbinding activities of 6 C-terminal deletion mutants of ENOl.
  • the binding epitope of EN 10 mAb is locatedbetween amino residue number 296 and 336of human ENOl protein.
  • the large portion deletion of ENOl to determine the binding region of EN10 mAb was performed as described in Example 12.
  • FIG. 13 A depicts the crystal structure and surface-expose amino acid residues between amino number 296 and 336 of human ENOl. The structure prediction was describedin Example 13.
  • FIG. 13B shows the 12% SDS PAGE of 11 alanine scanning mutantproteins of ENOl purified from E.coli. The detailedproceduresfor the purification of ENOl mutation proteinsaredescribed in Example 13.
  • FIG. 13C shows the ENOl binding ELISA and K d values of 11 alanine scanning mutants against EN 10 mAb.
  • the result suggests thatsequencesof ENOl peptide 1,FD Q D D W G A W Q F TA (SEQ ID NO: 9), and peptide 2,K R I A K A V N EK.S (SEQ ID NO:10),located between amino residue number 296 and 336 of human ENOl are involved inENlO mAbbinding.
  • the alanine scanning was performed as described in Example 13.
  • FIG. 13D shows the sequences of ENOl peptide 1 (FD Q D D W G A W Q K
  • FIG. 14A shows a 3D structure of human ENO 1 protein.
  • FIG.14B depicts the deduced structure of EN 10 mAb Fab (shown in ribbon-helix, light chain grey, heavy chain black) in complex with the plasminogen binding region of human ENOl protein. Crystal structure prediction of ENOl protein and EN 10 mAb was performed as described in Example 14.
  • FIG. 15A shows an expression vector for the generation of mouse-human chimera of EN 10 mAb. The detailedproceduresfor thepurification of the EN 10 mAb chimera antibody aredescribed in Example 15.
  • FIG. 15B depicts results from using the chimera antibody to determine binding affinity and kinetic constants of EN10 mAb. Detailed procedures of chimera antibody expression, purification andK analysis were performed as described in Example 15.
  • FIG. 16 shows inhibitory effects of EN10 mAb on the lung tumor growth in the presence of complement.
  • the administration of EN10 mAb and the retardation of tumor growth by antibody treatment were performed as described in Example 16. Thedata show that the administration of EN10 mAb with complementtwice per week has anefficacy similar to that treated with asame dose of commercial drugErbituxTM in the CLl-5 xenograft mouse model.
  • FIGs. 17A, 17B, and 17C show results fromblockade of tumor spread of pancreatic cancers in spleen-liver metastatic colony formation assay by EN10 mAb.
  • the administration of EN10 mAb and the inhibition of metastatic tumorgrowth by antibody treatment were performed as described in Example 17.
  • Thestudy results indicate that the administration of 10 mpk (mg/kg)EN10 mAb twice per week decreases themetastatic tumor nodule numbers, tumor volumes, and tumor weights of mice compared to those of the same dose of control IgG inthe spleen-liver metastatic mouse model.
  • Standard techniques are used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques are performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001)), which is incorporated herein by reference.
  • An antagonist may be a polypeptide, nucleic acid, carbohydrate, lipid, small molecular weight compound, an oligonucleotide, an oligopeptide, interference RNA (RNAi), antisense, a recombinant protein, an antibody, or conjugates or fusion proteins thereof.
  • RNAi interference RNA
  • antisense approach see Opalinska J B, Gewirtz A M. (Sci STKE. 2003 Oct. 28; 2003 (206): pe47).
  • ENOl disease-related aberrant activation or expression of "ENOl" may be any abnormal, undesirable or pathological cell adhesion, for example tumor-related cell adhesion.
  • Cell adhesion-related diseases include, but are not limited to, non-solid tumors such as leukemia, or lymphoma, and also solid tumors such as melanoma, non-small cell lung cancer, hepatocellular (liver) carcinoma,, gastric, head and neck, hepatic system, stomach, breast, ovary, lung, lung, uterus, vulva, colorectum, and pancreas.
  • ENOl refers to the heterodimer enolase molecule consisting of an
  • antibody refers generally and broadly to immunoglobulins, autoantibodies, monoclonal antibodies, and polyclonal antibodies, as well as active fragments thereof.
  • the fragment may be active in that it binds to the cognate antigen, or it may be active in that it is biologically functional.
  • the antibodies of the invention may be chimeric, humanized, or human, using techniques known in the art.
  • the term "monoclonal antibody” refers to antibodies that are chemically and immunologically homogeneous, generally produced by hybridomas. See A Laboratory Manual, Harlow and Lane, eds., Cold Spring Harbor, N.Y. (1988).
  • polyclonal antibody refers to antibodies that are produced by more than one clone of antibody-synthesizing plasma cells (B-lymphocytes) in response to the same antigen. They are generally produced by the animal after it is immunized with the antigen.
  • chimeric antibody refers to antibodies that contain sequences from more than one source. For example, such antibodies may contain sequences from non-human sources that are then modified by introducing human sequences.
  • humanized antibody refers to an antibody in which minimal portions of a non-human antibody are introduced into an otherwise human antibody.
  • human antibody refers to an antibody in which substantially every part of the protein is substantially non-immunogenic in humans, with only minor sequence changes or variations.
  • alpha-enolase specific antibody refers to an antibody that has a high specificity for mammalian ENOl, but not to EN02 or EN03.
  • ENOl -specific antibody refers to an antibody that binds the alpha-enolase protein.
  • neutralizing when referring to a targeted binding agent, such as an antibody, relates to the ability of said targeted binding agent to eliminate, or significantly reduce, the activity of a target antigen. Accordingly, a “neutralizing" ENOl antibody is capable of eliminating or significantly reducing the activity of ENOl.
  • a neutralizing ENOl antibody may, for example, act by blocking the binding of ENOl to the plasminogen. By blocking this binding, the plasminogen mediated cell dissociation is significantly, or completely, eliminated. Ideally, a neutralizing antibody against ENOl enhances cell adhesion.
  • polypeptide is used herein as a generic term to refer to native protein, fragments, or analogs of a polypeptide sequence.
  • native protein, fragments, and analogs are species of the polypeptide genus.
  • Preferred polypeptides in accordance with the invention comprise the human heavy chain immunoglobulin molecules and the human kappa light chain immunoglobulin molecules, as well as antibody molecules formed by combinations comprising the heavy chain immunoglobulin molecules with light chain immunoglobulin molecules, such as the kappa or lambda light chain immunoglobulin molecules, and vice versa, as well as fragments and analogs thereof.
  • Preferred polypeptides in accordance with the invention may also comprise solely the human heavy chain immunoglobulin molecules or fragments thereof.
  • polynucleotide as referred to herein means a polymeric form of nucleotides of at least 10 bases in length, either ribonucleotides or deoxynucleotides or a modified form of either type of nucleotide, or RNA-DNA hetero-duplexes.
  • the term includes single and double stranded forms of DNA.
  • CDR region or "CDR” is intended to indicate the hypervariable regions of the heavy or light chains of the immunoglobulin as defined by Kabat et al., 1991 ( abat, E. A. et al., (1991) Sequences of Proteins of Immunological Interest, 5th Edition. US Department of Health and Human Services, Public Service, NIH, Washington), and later editions.
  • An antibody typically contains 3 heavy chain CDRs and 3 light chain CDRs.
  • the term CDR or CDRs is used here in order to indicate, according to the case, one of these regions or several, or even the whole, of these regions which contain the majority of the amino acid residues responsible for the binding by affinity of the antibody for the antigen or the epitope which it recognizes.
  • HCDR3 has a greater size variability (greater diversity essentially due to the mechanisms of arrangement of the genes which give rise to it). It may be as short as 2 amino acids, although the longest size known is 26. CDR length may also vary according to the length that can be accommodated by the particular underlying framework. Functionally, HCDR3 plays a role in part in the determination of the specificity of the antibody (Segal et al, PNAS, 71:4298-4302, 1974).
  • a "set of CDRs" referred to herein comprises CDR1, CDR2 and
  • HCDR refers to HCDR1, HCDR2 and HCDR3
  • LCDR refers to LCDR1, LCDR2 and LCDR3
  • LCDR refers to a variable light chain CDR
  • a "set of CDRs" includes HCDRs and LCDRs.
  • polynucleotide sequence is homologous (i.e., is identical, not strictly evolutionarily related) to all or a portion of a reference polynucleotide sequence, or that a polypeptide sequence is identical to a reference polypeptide sequence.
  • the term “complementary to” is used herein to mean that the complementary sequence is homologous to all or a portion of a reference polynucleotide sequence.
  • the nucleotide sequence "TATAC” corresponds to a reference sequence “TATAC” and is complementary to a reference sequence "GTATA.”
  • sequence identity means that two polynucleotide or amino acid sequences are identical (i.e., on a nucleotide -by-nucleotide or residue-by-residue basis) over the comparison window.
  • percentage of sequence identity is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I) or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
  • the identical nucleic acid base e.g., A, T, C, G, U, or I
  • substantially identical denotes a characteristic of a polynucleotide or amino acid sequence, wherein the polynucleotide or amino acid comprises a sequence that has at least 85 percent sequence identity, preferably at least 90 to 95 percent sequence identity, more preferably at least 99 percent sequence identity, as compared to a reference sequence over a comparison window of at least 18 nucleotide (6 amino acid) positions, frequently over a window of at least 24-48 nucleotide (8-16 amino acid) positions, wherein the percentage of sequence identity is calculated by comparing the reference sequence to the sequence which may include deletions or additions which total 20 percent or less of the reference sequence over the comparison window.
  • the reference sequence may be a subset of a larger sequence.
  • the twenty conventional amino acids and their abbreviations follow conventional usage.
  • Stereoisomers e.g., D-amino acids
  • unnatural amino acids such as , ⁇ -disubstituted amino acids, N-alkyl amino acids, lactic acid, and other unconventional amino acids may also be suitable components for polypeptides of the present invention.
  • Examples of unconventional amino acids include: 4-hydroxyproline, ⁇ -carboxyglutamate, ⁇ - ⁇ , ⁇ , ⁇ -trimethyllysine, ⁇ - ⁇ -acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, ⁇ - ⁇ -methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline).
  • the left-hand direction is the amino terminal direction and the right-hand direction is the carboxy-terminal direction, in accordance with standard usage and convention.
  • the left-hand end of single-stranded polynucleotide sequences is the 5' end; the left-hand direction of double-stranded polynucleotide sequences is referred to as the 5' direction.
  • the direction of 5' to 3' addition of nascent RNA transcripts is referred to as the transcription direction; sequence regions on the DNA strand having the same sequence as the RNA and which are 5' to the 5' end of the RNA transcript are referred to as "upstream sequences"; sequence regions on the DNA strand having the same sequence as the RNA and which are 3' to the 3' end of the RNA transcript are referred to as "downstream sequences".
  • the term "substantial identity” means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 80 percent sequence identity, preferably at least 90 percent sequence identity, more preferably at least 95 percent sequence identity, and most preferably at least 99 percent sequence identity. Preferably, residue positions that are not identical differ by conservative amino acid substitutions.
  • amino acid sequences of antibodies or immunoglobulin molecules are contemplated as being encompassed by the present invention, providing that the variations in the amino acid sequence maintain at least about 75%, more preferably at least 80%, 90%, 95%, and most preferably about 99% sequence identity to the antibodies or immunoglobulin molecules described herein.
  • conservative amino acid replacements are contemplated.
  • Whether an amino acid change results in a functional peptide can readily be determined by assaying the specific activity of the polypeptide derivative. Assays are described in detail herein. Fragments or analogs of antibodies or immunoglobulin molecules can be readily prepared by those of ordinary skill in the art. Preferred amino- and carboxyl-termini of fragments or analogs occur near boundaries of functional domains. Structural and functional domains can be identified by comparison of the nucleotide and/or amino acid sequence data to public or proprietary sequence databases. Methods to identify protein sequences that fold into a known three-dimensional structure are known. Bowie et al., (1991) Science 253:164. Thus, the foregoing examples demonstrate that those of skill in the art can recognize sequence motifs and structural conformations that may be used to define structural and functional domains in accordance with the antibodies described herein.
  • a further aspect of the invention is a targeting binding agent or an antibody molecule comprising a VH domain that has at least about 60, 70, 80, 85, 90, 95, 98 or about 99% amino acid sequence identity with a VH domain of any of antibodies shown in sequences 1, the appended sequence listing, an antibody described herein, or with an HCDR (e.g., HCDR1, HCDR2, or HCDR3) shown in sequences 1.
  • HCDR e.g., HCDR1, HCDR2, or HCDR3
  • the targeting binding agent or antibody molecule may optionally also comprise a VL domain that has at least about 60, 70, 80, 85, 90, 95, 98 or about 99% amino acid sequence identity with a VL domain any of antibodies shown in sequences 2, the appended sequence listing, an antibody described herein, or with an LCDR (e.g., LCDR1, LCDR2, or LCDR3) shown in sequences 2.
  • LCDR e.g., LCDR1, LCDR2, or LCDR3
  • Algorithms that can be used to calculate % identity of two amino acid sequences comprise e.g. BLAST (Altschul et al., (1990) J. Mol. Biol.
  • the targeting binding agent or antibody that shares amino acid sequence identity as describes above exhibits substantially the same activity as the antibodies referenced.
  • substantially the same activity comprises at least one activity that differed from the activity of the references antibodies by no more that about 50%, 40%, 30%, 20%, 10%, 5%, 2%, 1% or less.
  • An antigen binding site is generally formed by the variable heavy (VH) and variable light (VL) immunoglobulin domains, with the antigen-binding interface formed by six surface polypeptide loops, termed complimentarity determining regions (CDRs). There are three CDRs in each VH (HCDR1, HCDR2, HCDR3) and in each VL (LCDR1, LCDR2, LCDR3), together with framework regions (FRs).
  • CDRs complimentarity determining regions
  • HCDR1, HCDR2, HCDR3 three CDRs in each VH
  • LCDR1, LCDR2, LCDR3 LCDR1, LCDR2, LCDR3
  • FRs framework regions
  • a VH domain is paired with a VL domain to provide an antibody antigen-binding site, although a VH or VL domain alone may be used to bind antigen.
  • the VH domain (e.g. from sequence 1) may be paired with the VL domain (e.g.
  • An antigen binding site may comprise a set of H and/or L CDRs of the parent antibody or any of antibodies in sequences 1 and 2with as many as twenty, sixteen, ten, nine or fewer, e.g. one, two, three, four or five, amino acid additions, substitutions, deletions, and/or insertions within the disclosed set of H and/or L CDRs. Such modifications may potentially be made at any residue within the set of CDRs.
  • Preferred amino acid substitutions are those which: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter binding affinities, and (4) confer or modify other physicochemical or functional properties of such analogs.
  • Analogs can include various mutant proteins of a sequence other than the naturally-occurring peptide sequence.
  • single or multiple amino acid substitutions may be made in the naturally-occurring sequence (preferably in the portion of the polypeptide outside the domain(s) forming intermolecular contacts.
  • a conservative amino acid substitution should not substantially change the structural characteristics of the parent sequence (e.g., a replacement amino acid should not tend to break a helix that occurs in the parent sequence, or disrupt other types of secondary structure that characterizes the parent sequence).
  • a further aspect of the invention is an antibody molecule comprising a VH domain that has at least about 60, 70, 80, 85, 90, 95, 98 or about 99% amino acid sequence identity with a VH domain of any of antibodies listed in sequence 1, the appended sequence listing or described herein, or with an HCDR (e.g., HCDR1, HCDR2, or HCDR3) shown in sequences 1.
  • HCDR e.g., HCDR1, HCDR2, or HCDR3
  • the antibody molecule may optionally also comprise a VL domain that has at least 60, 70, 80, 85, 90, 95, 98 or 99% amino acid sequence identity with a VL domain of any of the antibodies shown in sequences 2, the appended sequence listing or described herein, or with an LCDR (e.g., LCDRl, LCDR2, or LCDR3) shown in sequence 2.
  • LCDR e.g., LCDRl, LCDR2, or LCDR3
  • Algorithms that can be used to calculate % identity of two amino acid sequences comprise e.g. BLAST, FASTA, or the Smith- Waterman algorithm.
  • Variants of the VH and VL domains and CDRs of the present invention including those for which amino acid sequences are set out herein, and which can be employed in targeting agents and antibodies for human ENOl protein can be obtained by means of methods of sequence alteration or mutation and screening for antigen targeting with desired characteristics.
  • desired characteristics include but are not limited to: increased binding affinity for antigen relative to known antibodies which are specific for the antigen; increased neutralization of an antigen activity relative to known antibodies which are specific for the antigen if the activity is known; specified competitive ability with a known antibody or ligand to the antigen at a specific molar ratio; ability to immunoprecipitate complex; ability to bind to a specified epitope; linear epitope, e.g.
  • peptide sequence identified using peptide-binding scan as described herein e.g. using peptides screened in linear and/or constrained conformation; conformational epitope, formed by non-continuous residues; ability to modulate a new biological activity of human ENOl protein or downstream molecule.
  • peptide-binding scan as described herein, e.g. using peptides screened in linear and/or constrained conformation; conformational epitope, formed by non-continuous residues; ability to modulate a new biological activity of human ENOl protein or downstream molecule.
  • a further aspect of the present invention relates to a targeted binding agent(i.e. an antibody) including those for which amino acid sequencesthat binds to the epitope peptide comprising amino acid sequence that has at least about 60, 70, 80, 85, 90, or about 92% amino acid sequence identity listed in sequences 9 or lOon human ENOl protein and can be used to treat an human ENOl protein disease or disorder.
  • a targeted binding agent i.e. an antibody
  • amino acid sequencesthat binds to the epitope peptide comprising amino acid sequence that has at least about 60, 70, 80, 85, 90, or about 92% amino acid sequence identity listed in sequences 9 or lOon human ENOl protein and can be used to treat an human ENOl protein disease or disorder.
  • a human ENOl protein-related disease or disorder can be any condition arising due to the aberrant activation or expression of human ENOl protein.
  • the human ENOl protein-related disease is a neoplastic disease such as non-small cell lung cancer, hepatocellular (liver) carcinoma, gastric (stomach) cancer, breast cancer, pancreatic duct adenocarcinoma.
  • a neoplastic disease such as non-small cell lung cancer, hepatocellular (liver) carcinoma, gastric (stomach) cancer, breast cancer, pancreatic duct adenocarcinoma.
  • An antibody antigen-binding site composed of a VH domain and a VL domain is typically formed by six loops of polypeptide: three from the light chain variable domain (VL) and three from the heavy chain variable domain (VH).
  • VL light chain variable domain
  • VH heavy chain variable domain
  • Analysis of antibodies of known atomic structure has elucidated relationships between the sequence and three-dimensional structure of antibody combining sites. These relationships imply that, except for the third region (loop) in VH domains, binding site loops have one of a small number of main-chain conformations: canonical structures.
  • the canonical structure formed in a particular loop has been shown to be determined by its size and the presence of certain residues at key sites in both the loop and in framework regions.
  • Variant sequences may be made, with substitutions that may or may not be predicted to have a minimal or beneficial effect on activity, and tested for ability to bind and/or neutralize and/or for any other desired property.
  • Variable domain amino acid sequence variants of any of the VH and VL domains whose sequences are specifically disclosed herein may be employed in accordance with the present invention, as discussed.
  • polypeptide fragment refers to a polypeptide that has an amino-terminal and/or carboxy-terminal deletion, but where the remaining amino acid sequence is identical to the corresponding positions in the naturally-occurring sequence deduced, for example, from a full-length cDNA sequence. Fragments typically are at least about 5, 6, 8 or 10 amino acids long, preferably at least about 14 amino acids long, more preferably at least about 20 amino acids long, usually at least about 50 amino acids long, and even more preferably at least about 70 amino acids long.
  • analog refers to polypeptides which are comprised of a segment of at least about 25 amino acids that has substantial identity to a portion of a deduced amino acid sequence and which has at least one of the following properties: (1) specific binding to human ENOl protein under suitable binding conditions, (2) ability to block appropriate ligand/ ENOl protein binding, or (3) ability to inhibit ENOl protein activity.
  • polypeptide analogs comprise a conservative amino acid substitution (or addition or deletion) with respect to the naturally-occurring sequence.
  • Analogs typically are at least 20 amino acids long, preferably at least 50 amino acids long or longer, and can often be as long as a full-length naturally-occurring polypeptide.
  • a “targeted binding agent” is an agent, e.g. antibody, or binding fragment thereof, that preferentially binds to a target site.
  • the targeted binding agent is specific for only one target site. In other embodiments, the targeted binding agent is specific for more than one target site.
  • the targeted binding agent may be a monoclonal antibody and the target site may be an epitope.
  • a targeted binding agent may comprise at least one antigen binding domain of an antibody, wherein said domain is fused or contained within a heterologous protein.
  • An antibody may be oligoclonal, a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a CDR-grafted antibody, a multi-specific antibody, a bi-specific antibody, a catalytic antibody, a chimeric antibody, a humanized antibody, a fully human antibody, an anti-idiotypic antibody and antibodies that can be labeled in soluble or bound form as well as fragments, variants or derivatives thereof, either alone or in combination with other amino acid sequences provided by known techniques.
  • An antibody may be from any species.
  • antibody also includes binding fragments of the antibodies of the invention; exemplary fragments include Fv, Fab, Fab', single stranded antibody (svFC), dimeric variable region (Diabody) and disulfide stabilized variable region (dsFv).
  • Binding fragments include Fab, Fab', F(ab') 2 , Fv, and single-chain antibodies.
  • Fab antigen-binding fragments
  • Fc antigen-binding fragment
  • Digestion of antibodies with the enzyme, pepsin results in the a F(ab') 2 fragment in which the two arms of the antibody molecule remain linked and comprise two-antigen binding sites.
  • the F(ab') 2 fragment has the ability to crosslink antigen.
  • Fv when used herein refers to the minimum fragment of an antibody that retains both antigen-recognition and antigen-binding sites.
  • Fab when used herein refers to a fragment of an antibody that comprises the constant domain of the light chain and the CHI domain of the heavy chain.
  • the term “mAb” refers to monoclonal antibody.
  • epitopic determinants includes any protein determinant capable of specific binding to an immunoglobulin or T-cell receptor.
  • Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and may, but not always, have specific three-dimensional structural characteristics, as well as specific charge characteristics.
  • An antibody is said to specifically bind an antigen when the dissociation constant is ⁇ 1 micro M, preferably ⁇ 100 nM, and most preferably ⁇ 10 nM.
  • anENOl polypeptide refers to a portion of anENOl polypeptide that has a biological or an immunological activity of a native ENOl polypeptide.
  • Biological when used herein refers to a biological function that results from the activity of the native ENOl polypeptide.
  • a preferred ENOl biological activity includes, for example, ENOl induced the plasminogen activity.
  • mammal when used herein refers to any animal that is considered a mammal. Preferably, the mammal is human.
  • subject includes human and veterinary subjects.
  • pharmaceutical agent or drug refers to a chemical compound or composition capable of inducing a desired therapeutic effect when properly administered to a patient.
  • Other chemistry terms herein are used according to conventional usage in the art, as exemplified by The McGraw-Hill Dictionary of Chemical Terms (Parker, S., Ed., McGraw-Hill, San Francisco (1985)).
  • substantially pure means an object species is the predominant species present (i.e., on a molar basis it is more abundant than any other individual species in the composition), and preferably a substantially purified fraction is a composition wherein the object species comprises at least about 50 percent (on a molar basis) of all macromolecular species present. Generally, a substantially pure composition will comprise more than about 80 percent of all macromolecular species present in the composition, more preferably more than about 85%, 90%, 95%, and 99%. Most preferably, the object species is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species.
  • monitoring refers to the process of detecting and/or observing the development of cancer by determining the abundance of ENOl protein in cancer cells.
  • Methods for determining the abundance of ENOl include, but are not limited to, measuring the binding of ENOl proteins and ENOl -specific antibodies, Western blotting, flow cytometry, immunohistochemistry (IHC), RT-PCR, and/or microarray analysis.
  • Anti-ENOl antibody inhibits the invasion capacity of human lung cancer Cells
  • An anti-GST mouse polyclonal antibody was used as a negative control group.
  • Two chambers were separated by a micropore filter (12 micrometer pore size) coated with matrigel. After the incubation period, the matrigel-coated filters were stained and the number of cells invading into the matrigel-coated filter was quantified under a microscope. The study was repeated three times. Data are presented as mean ⁇ SD. Thetest was used to compare activity between each group. The P values ⁇ 0.05 were considered statistically significant.
  • ENOl polyclonal antibody attenuates the tissue invasion capability of CL1-5F4 cells in lung.
  • highly-invasive CL1-5F4 cells with a luciferase reporter gene (l xlO 6 cells/mouse) were intravenously injected into NOD-SCID mice,together with an ENOl antibody or a control (CTL) antiserum twice per week.
  • CTL control
  • the lung metastatic colony formation was monitored 12 and 19 days after tumor injection using an IVIS system.
  • the mouse injected with CL1-5F4 cells with knockdown of ENOl gene was used as a negative control. After the mice were scarified ondays 12 and 19, the metastatic colonies and tumor sizes were visualized and counted.
  • ENOl antibody can attenuate the invasion and metastasis of cancer cells. Therefore, ENOl antibody can potentially be developedas a therapeutic antibody.
  • BALB/c mice were primed with purified recombinant human ENOl antigen (50 ⁇ g/mouse), followed by boosting the ENOl humoral responses with an emulsified CpGadjuvant.
  • the splenocytes were harvested, fused with Fo cells, and then diluted according to a standard protocol.
  • the hybridoma cells secreting monoclonal antibodies that recognize the ENOl antigen were selected by an antigen-based ELISA.
  • the selected clones were then verified by the binding activity of the antibodies to the cell-surface ENOl on CL1-5F4 lung adenocarcinoma cells, using intact whole cell staining in flow cytometric analysis, as described in Example 1.
  • U937 cells were treated with EN10 mAb ranging from 5 microgram/ml to 50 microgram/ml in a similar manner.
  • EN10 mAb recognizes LSP-Induced surfaceENOl in lymphoma Cells
  • a human lymphoma U937 cell line was grown in RPMI containing 10% FCS. Cells were treated with 1 microgram/ml of LPS for 6 hours to induce ENOl protein expression on the cell surface.
  • the intact whole cells were stained with or without EN10 mAb (1 :300 dilution), visualized with FITC-conjugated goat anti mouse IgG (Jackson Lab), and analyzed with FACScan flow cytometer (Becton Dickinson). ENOl expression is measured by the resulting fluorescence intensity.
  • EN10 mAb inhibits the dissociation of CL-5 cells from collagen and fibronectin
  • ENOl takes part in the cell dissociation pathway in the extracellular matrix.
  • One microgram/ml of fibronectin and 10 microgram/ml of collagen were, respectively,coated on a non-treated ELISA plate overnig ht.CLl-5 cells (4xl0 4 cells) were seeded on the plate, and 0, 6.25, 12.5, 25, and 50 microgram/ml of EN10 mAb, respectively, were added to 200 micro L of DMEM containing 10% FCS.Furthermore,10 microgram/mL Glu-plasminogen and 2nM tPA were added. Cells were incubated at 37°C for 24 hours and washed with PBS twice.
  • Results from these experiments are shown in FIG.10B and FIG. IOC.
  • the data indicate that cell numbers are directly proportional to the concentrations of treated EN10 mAbin both extracellular matrices when the medium contains the ENOl receptor down-stream proteases plasminogen and tPA.There are significant difference between 50 microgram EN10 mAb treated group andthe control IgG group (P ⁇ 0.05) in both extracellular matrix studies.
  • ENOl is involved in the dissociation pathway of CLl-5 cells from extracellular matrixes, presumably by enhancing the plasmin and tPA protease activity.
  • ENIO mAb functioningas an antagonist of ENOl, blocks the receptor activity of ENOl, resulting in the inhibition ofplasmin and tPA activation and, therefore, inhibits the dissociation activity of CLl-5 cells from extracellular matrixes and invasion.
  • the hybridoma was cultured in a RPMI medium (manufactured by Gibco) containing 10% FCS. After the cell number reached about 10> 10 6 /ml, the cells were collected by centrifugation, and then TRIzol® (manufactured by Invitrogen) was added to extract total RNA in accordance the instruction manual. Cloning f the variable region of the antibody cDNAs was performed using a mouse Ig-primer set (manufactured byNovagen) in accordance with the attached instruction manual.
  • the 1st strand cDNA was prepared using 5microgram of the total RNA as a template. Five microgram of total hybridoma RNA, 1. microL of 50ng/microL of random primers, and ImicroL of lOmM dNTP were mixed, and DEPC-treated water was added to lOmicroL in a 200 1 PCR tube. The reaction mixture was incubated at 65 °C for 5 min, and then placed on ice for at least 1 minute.
  • a reaction solution having a composition of 5 microLof cDNA, 5 microL of 10x reaction Buffer, 1 microL of lOmM dNTP mix, ImicroLof 2.5 unit Taq polymerase, and ImicroL of forward primer 1 andl microL of reverse primer 2 provided bythe primer set was prepared in a final volume of 50 microL with double distilled water and subjected to PCR.
  • the framework regions (FR1, FR2, FR3, and FR4) and CDRs (HCDR1(SEQ ID NO: 3), HCDR2(SEQ ID NO: 4), and HCDR3(SEQ ID NO: 5) are indicated.
  • FIG. 11B depicts the variable light chain region amino acid sequences of EN10 mAb (SEQ ID NO: 2).
  • the framework regions FR1, FR2, FR3, and FR4
  • CDRs LCDR1(SEQ ID NO: 6), LCDR2(SEQ ID NO: 7), and LCDR3(SEQ ID NO: 8) are indicated.
  • the other set of primers having the sequencesof5 ' -GGATCCTATCTATTCTC AAGATCC ATGCC-3 ' (SEQ ID NO: 16) and 5 ' -CTCGAGGTCATGGTGTCTC ATCGTTCGCTCGAG-3 ' (SEQ ID NO: 17), was used to amplify adeletion ⁇ 297-434 mutant.
  • a reaction solution having a composition of ImicroL of 1 : 1000 dilution of template DNA about O.lng, 5 microL of 10* reaction buffer, ImicroL of lOmM dNTP mix, 1 microL of 2.5 unit Ta polymerase , ImicroL of the forward primer, andlmicroLof the reverse primer was prepared in a final volume of 50 microLwith double distilled water and subjected to PCR.
  • reaction solution was subjected to 2% agarose gel electrophoresis to analyze the reaction products. Reaction products with the correct molecular weights were ligated into a pCR 2.1-TOPO vector (manufactured by Invitrogen) for subcloning in accordance with the attached instruction manual.
  • M13 forward (5'-GTAAACAACGACGGCGAG ⁇ 3'(SEQ ID NO: 11) and M13 reverse (5'- CAG GAAACA GCT ATG AC--3 * (SEQ ID NO: 12)) primers were then used to determine the nucleotide sequence. Every mutation clone with the correctsequences was digested with restriction enzymes BamRl andA3 ⁇ 4oI,and the digestion products weresubjected to 2% agarose gel electrophoresis. The insertion fragment of each mutant was cut from the agarose gel and purified with aGene Clean Kit in accordance with the attached instruction manual provided by the manufacturer(BIOlOl).
  • the BamRl and J3 ⁇ 4oIDNA fragment of each mutant was ligated to theBamRl and Xhol sites of mE.coli expression vectorpTRC His A (Invitrogen).
  • the resulting plasmid was transformed into E.co/ BL21 Rosseta.
  • the ENOl mutation protein was expressed in E.coli by IPTG induction and purified by Ni-agarose in accordance with the attached instruction manual provided by the manufacturer (Qiagen) after the bacteria pellet was sonicated. The purity of each mutant was analyzed by 12% SDS PAGE.To determine the binding activity of each mutant protein, 400ng of human ENOl protein was coated on a 96-well ELISA plate and plate was washed by PBS.
  • AriE.coli expression plasmidpTRC-HIS ENOl containing human ENOl cDNA gene was used as a template to amplify the ENOl deletion mutants.
  • SEQ ID NO: 16 were used as the forward primer to amplify deletion mutants 296-434, 316-434, 336-434, 376-434 and 396-434 with the SEQ ID NO:18, SEQ ID:19, SEQ ID:20, SEQ ID:2l, SEQ ID:22, and SEQ ID:23 primers, respectively.
  • a reaction solution having a composition of ImicroL of 1 :1000 dilution of template DNA about O.lng, 5 microL of 10x reaction buffer, 1 microL of lOmM dNTP mix, 1 microLof 2.5 unit Taq polymerase, ImicroL of forward primer, and 1 microLof reverse primer was prepared in a final volume of 50 microL with double distilled water and subjected to PCR.
  • a cycle of 94degree C for 10 minutes was used.
  • a cycle of 94degreeC for one minute, 52degreeC for one minute, and 72 degree C for 1 minute was repeated 35 times, and the reaction was incubated at 72degreeC for 10 more minutes.
  • reaction solution was subjected to 2%> agarose gel electrophoresis to analyze the reaction products.
  • Reaction products with the correct molecular weights were ligated into a pCR 2.1-TOPO vector (manufactured by Invitrogen) for subcloning in accordance with the attached instruction manual.
  • Ml 3 forward (5'-GTAAACAACGACGGCGAG ⁇ 3'(SEQ ID NO: 11)
  • M13 reverse (5'-CAGGAA AC AGCT ATG AC--3 ' (SEQ ID NO: 12) primers were then used to determine the nucleotide sequence.
  • Every mutation clone with the correct sequence was digested with restriction enzymes BamHl and Xhol and the digestion product was subjected to 2% agarose gel electrophoresis.
  • the DNA fragment of each mutant was isolated from the agarose gel and purified with a Gene Clean Kit in accordance with the attached instruction manual provided by the manufacturer (BIO 101).
  • the BamHl and Xhol DNA fragment of each mutant was ligatedinto theBamHl and Xhol sites of anE.c /z ' expression vector pTRC His A (Invitrogen).
  • the resulting plasmid was transformed into E.coliB ll Rosseta.
  • the ENOl mutation protein was expressed in E.coli y IPTG induction and purified by Ni-agarose in accordance with the attached instruction manual provided by the manufacturer (Qiagen) after the bacteria pellet was sonicated. The purity of each mutant was analyzed by 12% SDS PAGE.
  • 400ng of human ENOl protein or mutant protein was coated on a 96-well ELISA plate and plate was washed by PBS. 10 microgram of EN 10 mAb was added and incubated at 37°C for 1 hours. After the binding complex was washed with PBS twice, agoat anti-mouse IgG conjugated with HPRT was added.After 1 hour incubation, TMB was added. The binding affinity was determined by the readings of OD 405. Each study was repeated three times. Data are presented as mean ⁇ SD. T-test was used to compare activity between each group. P values ⁇ 0.05 are considered statistically significant.
  • a reaction solution having a composition of 3 microL of template DNA about 30ng, 5 microL of 10x reaction buffer, 1 microL of lOmM dNTP mix, 1 microL of 2.5 unit pfu polymerase, 12.5 microL ofl25ng forward primer, and 12.5 microL of 125ng reverse primer was prepared in a final volume of 50 microL with double distilled water and subjected to PCR. A cycle of 95degreeCfor 10 minutes was used. Then, a cycle of 95degreeC for 30 seconds, 55degreeC for 30 seconds, and 68 degree C for 6 minute was repeated 16 times.
  • the ENOl mutation protein was expressed in E.coli by IPTG induction and purified by Ni-agarose in accordance with the attached instruction manual provided by the manufacturer (Qiagen) after the bacteria pellet was sonicated. The purity of each mutant protein was analyzed by 12% SDS PAGE.
  • Light binding refers to binding between a specific binding agent (e.g., an antibody, an scFv or Fab fragment) and a ligand/target (e.g., a peptide, protein, or cell) with a dissociation constant(K d ) of 10 nM or lower, preferably 1.0 nM or lower.
  • a specific binding agent e.g., an antibody, an scFv or Fab fragment
  • a ligand/target e.g., a peptide, protein, or cell
  • K d dissociation constant
  • Amino acid residuesW301 and K330 are importantfor the binding between ENOl protein and EN10 mAb. If these two amino acid residue are mutated to alanine, respectively, the binding activities of these two ENOlmutants to EN10 mAb are significantly compromised.
  • the second class of amino acid residues includesA309, E334, 335 andD300. If E334, K335 and D300are mutated to alanineorthe A309 is mutated to glycine,respectively, the binding activities of these ENOlmutants to EN 10 mAb are compromised.
  • the crystal structure of Enolase alpha was downloaded from protein data bank (pdb-entry: 2PSN).
  • the Fab fragment structure of EN10 mAb was built by thehomology modeling. The first step wasto find a template structure with high sequence similarity to the EN10 mAb.
  • the NCBI database www.ncbi.nlm.nih.gov.
  • the heavy-chain framework pdb-entry: 3DGG
  • the light chain framework pdb-entry: 1F6L
  • the antibody homology model of EN 10 mAb was constructed with the programs Accelrys Discovery Studio. ENOl and antibody structures were used for docking.
  • FIG.14A 3D structure of human ENOl protein is shown in FIG.14A. Amino acidresidues W301, K330,300D, E334, AND K335 are labeled. The critical residues in plasminogen binding site of ENOl protein published by Wang et al. are indicated(250-FFRSGKY 256).
  • FIG.14B shows that the binding complex of ENOl and EN10 mAb is predicted about 5 A to 15 A close to the plasminogen binding site of ENOl. The gap between plasminogen binding site and EN10 mAb is far less than 34 A, which is the molecular sized of human plasminogen.
  • Biacore analysis using a soluble ENOl protein to bind antibodies immobilized on CM5 chips was performed for EN10 mAb to estimate their affinity for soluble antigen.
  • the host cells for expression the FreeStyle293 cells (manufactured by Invitrogen) were used.
  • the vector was introduced into the host cells bylipofectamine 2000in accordance with the attached instruction manual (manufactured by Invitrogen.) About 2.5 microgram of the antibody expression vector was linearized by restriction enzymes, the gene was introduced into 4 l0 6 cells, and cells were inoculated to a6-well culture plate.
  • the agent corresponding to a selection marker of the expression vector was added, and cells were continuously cultured to form a stable pool.
  • a culturesupernatant containing human IgG antibody was prepared by the method described below.
  • the antibody-producing cells was acclimated in aFree styleTM293 Expression Medium (GIBCO).
  • Thecells were cultured in a tissue culture flask, and the culture supernatant was collected when the viable rate of the cells was 90%.
  • the collected supernatant was filtered through 10 micrometer and 0.2 micrometer filters (manufactured by Millpore) to remove contaminants.
  • the culture supernatant containing the antibody was affinity-purified using Protein A (manufactured by Millipore), PBS as an absorption buffer, and 20 mM sodium citrate buffer (pH 3.0) as an elution buffer.
  • the elution fractions were adjusted to around pH 6.0 by adding 50 mM sodium phosphate buffer (pH 7.0).
  • the prepared antibody solution was replaced with PBS using a dialysis membrane (10,000 MW cut, manufactured by Spectrum Laboratories) and filter-sterilized through a membrane filter (manufactured by Millpore) having a pore size of 0.22 micrometer to yield the purified antibody.
  • the concentration of the purified antibody was determined by measuring the absorbance at 280 nm and converting the measured value based on 1.45 optimal density equaling 1 mg/ml.
  • CM5 surface plasmon resonance
  • EDC N-ethyl-N'-(3 -dimethylaminopropyl)-carbodiimide hydrochloride
  • NHS N-hydroxysuccinimide
  • ChimeraEN 10 mAb was diluted with 10 mM sodium acetate, pH 4.8, into 5microgram/ml before injection at a flow rate of 20 micro L/minute to achieve approximately 100 response units (RU) of coupled protein followed by the injection of 1M ethanolamine to block unreacted groups.
  • the EN10 mAb has a goodaffinity with K d about 2.31 I ⁇ 0.003xl0 "10 mol/L and a potential for the further development.
  • a CLl-5 mouse xenograft model was performed.
  • CL1-5F4 lung adenocarcinoma cells (l xl0 6 cells/mouse; 5mice/group) were subcutaneously inoculated at Day 0.
  • the therapeutic procedure was performed 14 days after the tumor inoculation by administrating lOmpk (mg/Kg) of an isotype control (CTL), EN 10 mAb, or ErbituxTM antibody twice per week.
  • CTL isotype control
  • EN 10 mAb ErbituxTM antibody twice per week.
  • the rabbit baby serum was given 2h post-injection.
  • the tumor volume and bodyweight of each mouse was measured weekly. Data arerepresented as mean ⁇ SD for individual groups.
  • mice bearing CLl-5 are about 30 to 100 mm 3 , and there are no significant tumor size differences among the control group, EN 10 mAb, and Erbitux treatment groups after day 25.
  • the tumor of mice in the control group starts to grow exponentially, and there are no significant tumor growths in the EribituxTM and EN 10 mAb treatment mice.
  • the average tumor sizes of both EN 10 mAb and ErbituxTMtreatment groups aresignificantly smaller, as compared to that of the control group with a P value of 0.004 and 0.003, respectively.
  • There are no significant tumor size difference tween EN10 mAb and ErbituxTM treatment groups (P 0.41). This result indicates that EN10 mAb and Erbitux have the tumor growth inhibitionactivity on CLl-5 cells when combined with complement C3 in the mouse xenograft model and EN 10 mAb has a good efficacy as a reagent for cancer therapy.
  • Results fromExamples 2 and 3 suggest that administration of ENOl antibody can attenuate the invasion and metastasis of cancer cells and results from Example 16 indicates that ENOl mAb has efficacy ininhibiting the tumor growth when combined with complement.
  • a spleen-liver metastatic assay were performed. PDAC1 pancreatic adenocarcinoma cells (l xl0 6 /mouse; 5 mice/group) were injected into spleen. Then, 10 mpk of EN10 mAb and IgG control mice were administrated into each group by IP, respectively. The mice were scarified 6 weeks after tumor inoculation. The number of tumor nodules in livers and the weight of organs, including lungs, spleens, livers, pancreas, and kidneys, of each mouse were counted and measured, respectively. Data are represented as mean ⁇ SD for individual groups.
  • Results are shownin FIGs. 17 A, 17B, and 17C.
  • EN10 mAb uses its ENOl plasminogen receptor antagonistactivity to inhibitthe plasminogen activation, thereby inducing down regulation of protease activity on the cell surface, which in turn results in the inhibition of dissociation of cancer cells from extracellular matrix.
  • ENOl antibodies e.g., EN10 mAb
  • an antibody of the invention may also be used to diagnose a cancerous condition.
  • cancer cells express ENOl on cell surfaces, while normal cells do not. Therefore, one can detect antibody binding to cell surface ENOl as an indication of cancerous conditions.
  • a test sample may be reacted with an anti-ENOl antibody (e.g., EN10 mAb) and the binding of the antibody to the sample may be determined, such as with ELISA, fluorescence cell sorting, fluorescence microscope, or any suitable method known in the art. If the binding is detected or is above a threshold value (e.g., a reference value for a normal sample), then one may conclude that the sample contains cancer cells.
  • an anti-ENOl antibody e.g., EN10 mAb
  • a threshold value e.g., a reference value for a normal sample

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Abstract

The present invention discloses an anti-human alpha-enolase (ENO1) antibody, which can bind the peptides, comprising amino-acid sequence 296FD Q D D W G A W Q K F TA309 (SEQ ID: #9 ) and/or 326K R I A K A V N EK S336 (SEQ ID: #10 ) of human ENOl protein (GenBank: AAH50642.1), has a favorable binding activity (the binding affinity is around 2.19 x 10-10 mol/L) and a remarkable capability to inhibit the cell invasion and tumor metastasis of a varied of tumors. The recognized peptides and antibody of the invention are useful for diagnosis, prognosis, and treatment of cancers that have been reported to express cell-surface ENO1 such as including lung, breast, pancreas, liver, colorectal, prostate cancers and solid tumors.

Description

ALPHA-ENOLASE SPECIFIC ANTIBODIES AND METHODS OF USES IN
CANCER THERAPY
Field of The Invention
[0001] The present invention relates to methodsforgeneration and use ofantibodiesthat bind specifically to the human alpha-enolase protein (ENOl).
Background of The Invention
[0002] Tumors result from aberrant, unrestrained proliferation of a single cell, generating a clone of transformed cells. Tumor cells may express unique antigens that can be recognized by the immune system. Tumor-associated antigens includemutated oncogenes, mutated normal cellular proteins, aberrantly expressed cellular proteins, abnormal cell-surface proteins, and oncogenic viral proteins. The immune system views these tumor-associated antigens as non-self andean produce antibodies to eradicate these foreign antigen-bearing tumor cells, while sparing the healthy cells. Therefore, identification of immunogenic tumor-associated antigens may be used as targets for clinical prognostic or therapeutic applications in cancer treatment.
[0003] Certain malignancies may be identified by pleural effusion, which is excess fluid in the space between the lung and chest wall. Lung carcinoma, breast carcinoma, and lymphoma cause about 75% of all malignant pleural effusions. Malignant pleural effusion may be enriched with lymphocytic infiltrates and tumor cells. Tumor-associated immune complexes or autoantibodies, such as anti-p53, antinuclear, and anti-L-Myc antibodies, have been found in effusion fluids and are associated with poor prognosis. Several lung tumor-associated antigens have also been identified in malignant effusion, including, cytokeratin 19 fragments, neuron-specific enolase (EN02), squamous cell carcinoma antigen, and soluble HLA-I, etc.
[0004] Alpha-enolase (enolase- 1, ENOl) is a multiple functional protein, which was first found as a key enzyme of the glycolysis pathways. Under normal conditions, ENOl is expressed in the cytosol. However, ENOl is also found to express on the cell surfacesof many cancer cellsas a plasminogen receptor and on activated hematopoietic cells, such as neutrophils, lymphocytes and monocytes. It is known that the up-regulation of plasminogen receptor proteins can inducea cascade response of the urokinase plasminogen activationsystem (uPAS).
[0005] The urokinase plasminogen activator system (uPAS) consists of the urokinase plasminogen activator (uPA), its cognate receptor (uPAR) and two specific inhibitors, the plasminogen activator inhibitor 1 (PAI-1) and plasminogen activator inhibitor 2 (PAI-2). Urokinase plasminogen activator converts plasminogen proenzyme into an active serine protease, plasmin. Plasmin is involved in a number of tissue remodeling processes, such as basement membrane (BM) and extracellular matrix (ECM) remodeling, which is required in tumor progression and metastasis. In addition, it has been shown that the uPAS may be involved in the neoplastic evolution, affecting tumor angiogenesis, malignant cell proliferation, adhesion and migration, intra- vascularization, and growth at the metastatic site.
[0006] Specifically, activation of plasminogen can result in extracellular matrix degradations, which in turncan lead toincreased metastasis of cancer cells and infiltration of immune cells. In other words,EN01 expression on cancer cell surfaces as a plasminogen receptor can increase invasion activities of the cancer cells. Therefore, ENOl is a potential target for cancer therapy.
SUMMARY OF THE INVENTION
[0007] Embodiments of the invention relate to targeted binding agents that specifically bind human ENOl, thereby inhibiting ligand (e.g., plasminogen) binding to ENOl. By inhibiting binding of plasminogen to ENOl, targeted binding agents of the invention can inhibit plasminogen activation, leading to reduced degradation of extracellular matrix, which in turn prevents or reduces dissociation of cancer cells from the extracellular matrix. Therefore, targeted binding agents in accordance with embodiments of the invention can be used to inhibit tumor growth and metastasis. Mechanisms by which this can be achieved may include,but are not limited to, inhibition of binding of a ligand (such as plasminogen) to its receptor ENOl, or abrogation of inter-reactions between the receptor ENOl and its ligands, thereby reducing the effective concentration of ENOl.
[0008] In accordance with one embodiment of the invention, a targeted binding agent is anantibody that can bindto humanENOlto prevent its ligands (e.g., plasminogen) from binding to ENOl. Preventing binding of plasminogen to the receptor can preventplasminogenactivation. This results in the inhibition of the urokinase plasminogen activation system (uPAS) in the extracellular matrix of cancer cells.
[0009] In accordance with some embodiments of the invention, the antibody may bind ENOl with high affinities, such as with a Kdof less than 0.3nM.Such tight binding agents can inhibit ENOl with high efficiencies.
[0010] In accordance with some embodiments of the invention, a targeted binding agent is an antibody that can bind to human ENOl and inhibit induced plasmin activity on cancer cells with high efficiencies, such as 80%, 90%>, or 100% inhibition. The inhibition assays may be performed by inducing ENOl expression (hence, plasminogen activation) in a cancer cell (such asU937 human lymphomacells) by treatmentwith 10 microgram/mL lipopolysaccharide(LPS) for 5 hours. Inhibition of such induced plasmin activity may be assayed with an antibody at a suitable concentration. Using an antibody of the invention, such inhibition may be detectedat antibody concentrations as low as 20 microgram/ml or less.
[0011] In accordance withsome embodiments of the invention, a targeted binding agent is an antibody that can bind to human ENOl . Such an antibody may be used to inhibit the invasion activity of a cancer cell. For example, antibodies of the invention can inhibit greater than 50%, 60%, or 70% ofthe invasion activity of human non-small cell lung carcinoma CLl-5 cells at antibody concentrations as low as 50 microgram/ml or less.
[0012] In accordance with some embodiments of the invention,a targeted binding agent is an antibody that can bind to humanENOl to inhibit extracellular matrix degradation, thereby inhibiting cancer cell dissociation from the extracellular matrix. For example, an antibody of the invention can inhibit greater than 40%, 50%, or 60% of plasminogen mediated dissociation of CLl-5 cells from collagen or fibronectin at antibody concentrations as low as 50 microgram/ml or less.
[0013] In accordance with embodiments of the invention, a targeted binding agent
(i.e. an antibody) may comprise a heavy chain amino acid sequence having a complementarity determining region (CDR) comprising one of the CDR sequencesincludedin sequence 1.
[0014] In accordance with someembodiments of the inventions targeted binding agent (i.e. an antibody) may comprise a light chain amino acid sequence having a complementarity determining region (CDR) comprising one of the CDR sequences includedin sequence 2.
[0015] In accordance with someembodiments of the invention, a targeted binding agent is an antibody, which may bea monoclonal antibody or a polyclonal antibody.
[0016] In accordance with some embodiments of the invention,an antibodythat can bindhuman ENOlproteincomprises a light chain amino acid sequence comprising any one of LCDR1, LCDR2 or LCDR3 sequence included in sequence 2.
[0017] In accordance with some embodiments of the invention, an antibodythat can bind human ENOlproteincomprises a light chain amino acid sequence comprising any two of LCDR1, LCDR2 or LCDR3 sequence included in sequence 2(that is,LCDRl and LCDR2, LCDR1 and LCDR3 or LCDR2 and LCDR3).
[0018] In accordance with some embodiments of the invention, an antibodythat can bind human ENOlproteincomprisesa light chain amino acid sequencesthat comprisesLCDRl, LCDR2 and LCDR3 sequencesincluded in sequence 2. In accordance with certain embodiments of the invention,an antibody may be a humanized antibody ora fully human monoclonal antibody. [0019] In accordance with some embodiments of the invention,an antibodythat can bind human ENOl protein comprisesa heavy chain amino acid sequence comprising any one of HCDRl, HCDR2 or hCDR3 sequence includedin sequence 1.
[0020] In accordance with some embodiments of the invention, an antibodythat can bind human ENOl protein comprises a heavy chain amino acid sequence that comprises any two of HCDRl, HCDR2 or HCDR3 sequence included in n sequence 1 (that is,HCDRl and HCDR2, HCDRl and HCDR3 or HCDR2 and HCDR3).
[0021] In accordance with some embodiments of the invention, an antibodythat can bind human ENOl protein comprisesa heavy chain amino acid sequencethat comprisesHCDRl , HCDR2 and HCDR3 sequencesincluded insequence 1. In accordance with some embodiments of the invention, an antibody may be a humanized antibody or a fully human monoclonal antibody.
[0022] In accordance with some embodiments of the invention, an antibody that can bind human ENOl protein comprises a light chain amino acid sequence having a CDR comprising one of the CDR sequences includedin sequence 2. In accordance with some embodiments of the invention, an antibody can bind human ENOl protein and comprises a heavy chain amino acid sequence having a CDR comprising one of the sequences shown in sequences 1. In accordance with some embodiments of the invention, an antibody may be a humanized or a fully human monoclonal antibody.
[0023] In accordance with some embodiments of the invention, an antibodythat can bind human ENOl protein comprises a heavy chain amino acid sequence having one of the CDR sequences included in sequence 1 and a light chain amino acid sequence having one of the CDR sequences included in sequence 2. In accordance with some embodiments of the invention, an antibody may be a humanized or a fully human monoclonal antibody.
[0024] In accordance with some embodiments of the invention, a targeted binding agent (i.e. an antibody) can compete for binding of plasminogen to human ENOl protein.In accordance with some embodiments of the invention, said targeted binding agent comprises a heavy chain amino acid sequence having at least one of the CDR sequences included in sequence 1 and a light chain amino acid sequence having at least one of the CDR sequences included in sequence 2.
[0025] In accordance with some embodiments of the invention, a targeted binding agent can bind to an epitopecomprising the amino acid sequence296FD Q D D W G A W Q F TA309 (SEQ ID NO:9 ) or326 R I A K A V N EK S336 (SEQ ID NO:10 ) of human ENOl protein (GenBank: AAH50642.1). In accordance with some embodiments of the invention, said targeted binding agent comprises a heavy chain amino acid sequence having at least one of the CDR sequences included in sequence 1 and a light chain amino acid sequence having at least one of the CDR sequences included in sequence 2.
[0026] In accordance with some embodiments of the invention, a binding agent of the invention may comprise an antigen-binding site within a non-antibody molecule. For example, such a binding agent maycomprise one or more CDRs e.g. a set of CDRs in a non-antibody protein scaffold, as discussed further below.
[0027] Some embodiments of the invention relate to methods for assaying the level of human ENOl protein in a patient or a patient sample. A methodof the invention comprises contacting an anti-ENOl antibodywith a biological sample from a patient, and detecting the level of binding between said antibody and human ENOl protein in said sample. In more specific embodiments, the biological sample is blood or plasma.
[0028] Other embodiments of the invention relate to compositions comprising a targeted binding agent, which may include an antibody or a functional fragment thereof, and a pharmaceutically acceptable carrier.
[0029] Still further embodiments of the invention relate to methods for effectively treating a subject (e.g., human or animal) suffering from an ENOl disease or disorder. The method may include selecting a subject in need of a treatment for a neoplastic or non-neoplastic disease, and administering to the subject a therapeutically effective dose of an antibody (which may be a humanized or a fully human monoclonal antibody) that specifically binds to ENOl protein.
[0030] The antibody of the invention can be used to treat a human ENOl protein-relateddisease or disorder. A human ENOl protein related disease or disorder may be any condition arising from aberrant activation or expression of human ENOl protein. Examples of such diseases include where human ENOl protein aberrantly interacts with its ligands, thereby altering cell-adhesion or cell signaling properties. This alteration in cell adhesion or cell signaling properties can result in neoplastic diseases or some immune diseases.
[0031] Forexample, a human ENOl protein-related disease may be a neoplastic disease, such aslung, breast, pancreas, liver, colorectal, and prostate cancers.
[0032] In accordance withsome embodiments of the invention, a targeted binding agent(e.g., an antibody)can bind to the peptide comprising the amino acid sequence FD Q D D W G A W Q K F T308 (SEQ ID NO:9) or326 K R I A K A V N EK S336 (SEQ ID NO:10) of human ENOl protein (GenBank: AAH50642.1)and can be used to treat a human ENOl protein-related disease or disorder noted above.
[0033] Additional embodiments of the invention relate to methods for inhibiting
ENOl -induced cell dissociationfrom extracellular matrix of cancersin a subject. These methods may include selecting a subject (e.g., a human or an animal) in need of treatment for ENOl -induced cell dissociation, and administering to said subject a therapeutically effective dose of an antibody, wherein said antibody specifically binds toENOl. The antibody may be a humanized or fully human monoclonal antibody.
[0034] Further embodiments of the invention relate to the uses of an antibody in the preparation of a medicament for the treatment of an ENOl -related disease or disorder in a subject (e.g., a human or an animal), wherein said antibody specifically binds toENOl . The antibody may be a humanized or fully human monoclonal antibody.
[0035] In accordance with some embodiments of the invention, the targeted binding agents described herein can be used for the preparation of a medicament for the treatment of ENOl protein-induced cell dissociationfrom extracellular matrix in an animal, wherein said antibody specifically binds toENOl. The antibody may be a humanized or fully human monoclonal antibody.
[0036] Some embodiments of the invention described herein relate to monoclonal antibodiesthat bindhuman ENOland affect human ENOl functions. Other embodiments of the invention relate to anti ENOl antibody preparations with desirable properties for therapy applications. Such properties may include a high binding affinity for ENOl, the ability to neutralize ENOl activity in vitro and in vivo, and the ability to inhibit ENOl induced cell dissociation,growth and metastasis of tumors.
[0037] In some embodiments, the invention relates to anantibody that can bindto human ENOlwith very high affinity (i.e., low ¾). For example a human, rabbit, mouse, chimeric or humanized antibody that is capable of binding ENOl with a Kjless than about 10"5, 10~6, 10"7, 10~8, 10"9, 10"10 or about 10"ΠΜ, or any range or value therebetween. Affinity and/or avidity measurements can be performed using ELISA and/or BIACORE, as described herein or according to techniques known in the art.
[0038] It will be appreciated that embodiments of the invention are not limited to any particular form of an antibody or method of generation or production. For example, the anti-ENOl antibody may be a full-length antibody (e.g., having an intact human Fc region) or an antibody fragment (e.g., a Fab, Fab' or F(ab')2, FV or Dab (Dabs are the smallest functional binding units of human antibodies). In addition, the antibody may be manufactured from a hybridoma that secretes the antibody, or from a recombinantly produced cell that has been transformed or transfected with a gene or genes encoding the antibody.
[0039] Other embodiments of the invention relate to isolated nucleic acid molecules encoding any of the antibodies described herein, vectors having isolated nucleic acid molecules encoding anti-ENOl antibody or a host cell transformed with any of such nucleic acid molecules. [0040] In addition, some embodiments of the invention relate to a method for producing an anti-ENOl antibody by culturing host cells under conditions wherein a nucleic acid molecule is expressed to produce the antibody, followed by recovering the antibody. It should be realized that embodiments of the invention may also include any nucleic acid molecule which encodes an antibody or fragment of an antibody of the invention including nucleic acid sequences optimized for increasing yields of antibodies or fragments thereof when transfected into host cells for antibody production.
[0041] Further embodiments of the invention mayrelate to methodsfor producing high affinity antibodies to human ENOl by immunizing a mammal with human ENOl protein, a fragment thereof, and one or more orthologous sequences or fragments thereof.
[0042] Other embodiments relate to the generation and identification of isolated antibodies that can bind specifically to human ENOl . Inhibition of the biological activity of ENOl can be effected by these antibodies to prevent ENOl induced cell dissociation, invasion and other desired effects of cancers.
[0043] Other embodiments of the invention relate to pharmaceutical compositions having an effective amount of an anti-ENOl antibody. The composition may further comprise a pharmaceutically acceptable carrier or diluent. In yet other embodiments, the anti-ENOl antibody, or a fragment thereof, is conjugated to a therapeutic agent. The therapeutic agent can be, for example, a toxin or a radioisotope.
[0044] Yet other embodiments of the inventionrelate to methods for treating diseases or conditions associated with the expression of ENOl in a patient. The methods may include administering to a patient an effective amount of an anti-ENOl antibody. The anti-ENOl antibody can be administered alone, or can be administered in combination with additional antibodies or chemotherapeutic drug or radiation therapy. For example, a monoclonal, oligoclonal or polyclonal mixture of ENOl antibody that block cell dissociation can be administered in combination with a drug shown to inhibit tumor cell proliferation directly. The method can be performed in vivo and the patient is preferably a human patient. In a preferred embodiment, the method concerns the treatment of an ENOl -related disease or disorder including, but not limited to, neoplastic diseases, such as lung, breast, pancreas, liver, colorectal, prostate cancers and or solid tumors.
[0045] Some embodiments of the invention relate to a method for monitoring cancer development. The method may comprise determining the abundance of alpha-enolase proteins (ENOl) in a sample (e.g., cancer cells), wherein an increased level of ENOl correlates with cancer severity. In accordance with embodiments of the invention, the abundance may be determined by measuring binding of an ENOl specific antibody to the ENOl proteins. [0046] Some embodiments of the invention relate to a method for detecting cancer.
Such a method may comprise determining the abundance of ENOl -specific antibodies in serum samples, wherein a low level of ENOl -specific antibodies indicates the presence of a malignant tumor.
Brief Description of the Drawings
[0047] FIGs. 1A and IB show results from studies ofcell surface location of ENOl in malignant cells using flowcytometry (FIG. 1A) and immunostaining (FIG. IB), respectively.Detailed procedures were performed as described in Example l .The data showthat ENOl is located on cell surface in malignant cells (NHRI-L89 & CA926), but not in normal lung epithelial cells (NHBE & SAEC).
[0048] FIG. 2 shows the inhibition of invasion activity of highly-invasive CLl-5 lung adenocarcinoma cells by polyclonal antibody against cell-surface ENOl. Detailed procedures were performed as described in Example 2. The data show that administrating anti-ENOl polyclonal antibody attenuates the invasion of CLl-5.
[0049] FIG. 3 shows that apolyclonal antibody against human ENOl attenuates tissue invasion capability of CL1-5F4 cells in lung.The administration of antibody in an animal and the lung metastatic colony formation was monitored 12 and 19 days after tumor injection using an IVIS system. The detailed procedureswere performed as described in Example 3. The data show that ENOl polyclonal antibody inhibits the migration of CL1-5F4 to lung in vivo.
[0050] FIG. 4 shows the generation of anti-ENOl antibody hybridomas andverification of each monoclonal antibody clones by flow cytometry. The proceduresforthe immunization of mice and generation of hybridoma, and the production of each antibody and verification of antibody by flow cytometry were described in Example 4. The data show that all5 hybridoma antibodies recognize the surface ENOl on theCLl-5F4 lung adenocarcinoma cells.
[0051] FIG. 5A shows ENOl binding in ELISAof 5 antibodies isolated from acites of individual hybridomas. Ammonium sulfate purification, protein A column purification, and SDS-PAGE purification were performed as described in Example 5. The data in FIG. 5B show theKdvalues of 5 different anti human ENOl antibodies.
[0052] FIG. 6 shows the results of U937 fibrinolytic assay of 5 antibodies isolated from acites of individual hybridomas, respectively.The induction of ENOl expression by LPS in human U937 lymphoma cell line and the plasminactivity assay were performed as described in Example 6. These data also show that 5 different anti-ENOl antibodies have different inhibition activities againstplasminogen receptor of ENOl andthat the inhibition activitiescorrelate withthe ¾ values. [0053] FIG. 7 shows results of inhibition of invasion activities of CLl-5 cells treated with 5 different antibodies isolated from acites of individual hybridomas, respectively. The detailedprocedureswere described in Example 7. These data show that all 5 different anti-ENOl antibodies can inhibit the invasion activity of CLl-5 cells. Interestingly, even though clone 8 has aiQabout 7.2-foldshigher than that of clone 10, the inhibition of invasionactivity against CLl-5 cells byclone 8 is similarto that of clone 10.
[0054] FIG. 8 A shows results of invasion activities of CLl-5 cells treated with different concentrations of ENlOmAb isolated from hybridoma. Detailedprocedures were performed as described in Example 8. These data show thatENlOmAb antibody inhibits the invasion activity of CLl-5 in a dose-dependent manner.
[0055] FIG. 8B shows results of invasion activities of U937 cells treated with different concentrations of EN10 mAb isolated from hybridoma,after the surface ENOl expression of cells was induced by LPS. Detailedprocedures were performed as described in Example 8. These data show that the EN10 mAb inhibits the invasion activity of U937 cells in a dose-dependent manner.
[0056] FIG. 9 shows that EN10 mAb recognizes the cell surface ENOl on U937 cells treated with LPS. Detailed procedures were performed as described in Example 9.
[0057] FIG. 10A shows the adhesion activity of CLl-5 lung carcinoma cells to matrix proteins. The adhesion assay was performed as described in Example 10. These data show that CLl-5 cells have higher adhesion activities to collagen and fibronectin.
[0058] FIG. 10B shows results of inhibition of CLl-5 cell dissociationfromfibronectin treated with the EN10 mAb. The cell associated adhesion assay was performed as described in Example 10. These data show that the EN10 mAb inhibits the cell dissociation activity of CLl-5 from fibronectinin a dose-dependent manner.
[0059] FIG. IOC shows results of inhibition of CLl-5 cell dissociationfromcollagen treated with the EN10 mAb. The cell associated adhesion assay was performed as described in Example 10. These data show that the EN10 mAbinhibits the cell dissociation activity of CLl-5 fromcollagen ina dose-dependent manner.
[0060] FIG. 11A depicts the variable heavy chain region amino acid sequence of
EN10 mAb (SEQ ID NO: 1). The framework regions (FR1, FR2, FR3, and FR4) and CDRs (HCDR1, HCDR2, and HCDR3) are indicated. Cloning the EN10 mAb was performed as described in Example 11.
[0061] FIG. 1 IB depicts the variable light chain region amino acid sequence of EN10 mAb(SEQ ID NO: 2).The framework regions (FR1, FR2, FR3, and FR4) and CDRs (LCDR1, LCDR2, and LCDR3) are indicated. Cloning the EN10 mAb was performed as described in Example 11. [0062] FIG. 12A shows the EN10 mAbbinding activities of deletion mutants of
ENOl. The binding epitope of EN 10 mAb is locatedbetween the amino residue number 293 and 434 of human ENOl protein. The large portion deletion of ENOl to determine the binding region of EN10 mAb was performed as described in Example 12.
[0063] FIG. 12B shows the 12% SDS PAGE of 6 C-terminal deletion mutant protein of ENOl purified from E.coli. The detailedproceduresfor thepurification of ENOl deletion mutants aredescribed in Example 12.
[0064] FIG. 12C shows the EN 10 mAbbinding activities of 6 C-terminal deletion mutants of ENOl. The binding epitope of EN 10 mAb is locatedbetween amino residue number 296 and 336of human ENOl protein.The large portion deletion of ENOl to determine the binding region of EN10 mAb was performed as described in Example 12.
[0065] FIG. 13 A depicts the crystal structure and surface-expose amino acid residues between amino number 296 and 336 of human ENOl. The structure prediction was describedin Example 13.
[0066] FIG. 13B shows the 12% SDS PAGE of 11 alanine scanning mutantproteins of ENOl purified from E.coli. The detailedproceduresfor the purification of ENOl mutation proteinsaredescribed in Example 13.
[0067] FIG. 13C shows the ENOl binding ELISA and Kdvalues of 11 alanine scanning mutants against EN 10 mAb.The result suggests thatsequencesof ENOl peptide 1,FD Q D D W G A W Q F TA (SEQ ID NO: 9), and peptide 2,K R I A K A V N EK.S (SEQ ID NO:10),located between amino residue number 296 and 336 of human ENOl are involved inENlO mAbbinding.The alanine scanning was performed as described in Example 13.
[0068] FIG. 13D shows the sequences of ENOl peptide 1 (FD Q D D W G A W Q K
F TA (SEQ ID NO: 9)) and peptide 2 (K R I A K A V N EK S (SEQ ID NO:10))between amino residue number 296 and 336 of human ENOl (SEQ ID NO:49), which participate in human ENOl and EN10 mAbbinding.
[0069] FIG. 14A shows a 3D structure of human ENO 1 protein. FIG.14Bdepicts the deduced structure of EN 10 mAb Fab (shown in ribbon-helix, light chain grey, heavy chain black) in complex with the plasminogen binding region of human ENOl protein. Crystal structure prediction of ENOl protein and EN 10 mAb was performed as described in Example 14.
[0070] FIG. 15A shows an expression vector for the generation of mouse-human chimera of EN 10 mAb.The detailedproceduresfor thepurification of the EN 10 mAb chimera antibody aredescribed in Example 15. [0071] FIG. 15B depicts results from using the chimera antibody to determine binding affinity and kinetic constants of EN10 mAb. Detailed procedures of chimera antibody expression, purification andK analysis were performed as described in Example 15.
[0072] FIG. 16 shows inhibitory effects of EN10 mAb on the lung tumor growth in the presence of complement.The administration of EN10 mAb and the retardation of tumor growth by antibody treatment were performed as described in Example 16. Thedata show that the administration of EN10 mAb with complementtwice per week has anefficacy similar to that treated with asame dose of commercial drugErbitux™ in the CLl-5 xenograft mouse model.
[0073] FIGs. 17A, 17B, and 17C show results fromblockade of tumor spread of pancreatic cancers in spleen-liver metastatic colony formation assay by EN10 mAb.The administration of EN10 mAb and the inhibition of metastatic tumorgrowth by antibody treatment were performed as described in Example 17. Thestudy results indicate that the administration of 10 mpk (mg/kg)EN10 mAb twice per week decreases themetastatic tumor nodule numbers, tumor volumes, and tumor weights of mice compared to those of the same dose of control IgG inthe spleen-liver metastatic mouse model.
DEFINITIONS
[0074] Unless otherwise defined, scientific and technical terms used herein shall have the meanings that are commonly understood by those of ordinary skill in the art. Furthermore, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, and protein and oligo- or polynucleotide chemistry and hybridization described herein are those well-known and commonly used in the art.
[0075] Standard techniques are used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques are performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001)), which is incorporated herein by reference. The nomenclatures utilized in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.
[0076] As utilized in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings: The term "and/or" as used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example "A and/or B" is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein.
[0077] An antagonist may be a polypeptide, nucleic acid, carbohydrate, lipid, small molecular weight compound, an oligonucleotide, an oligopeptide, interference RNA (RNAi), antisense, a recombinant protein, an antibody, or conjugates or fusion proteins thereof. For a review of RNAi, see Milhavet O, Gary D S, Mattson M P. (Pharmacol Rev. 2003 December; 55(4):629-48. Review.) andfor antisense approach, see Opalinska J B, Gewirtz A M. (Sci STKE. 2003 Oct. 28; 2003 (206): pe47).
[0078] Disease-related aberrant activation or expression of "ENOl " may be any abnormal, undesirable or pathological cell adhesion, for example tumor-related cell adhesion. Cell adhesion-related diseases include, but are not limited to, non-solid tumors such as leukemia, or lymphoma, and also solid tumors such as melanoma, non-small cell lung cancer, hepatocellular (liver) carcinoma,, gastric, head and neck, hepatic system, stomach, breast, ovary, lung, lung, uterus, vulva, colorectum, and pancreas.
[0079] The term ENOl refers to the heterodimer enolase molecule consisting of an
ENOl and EN02 or EN03.
[0080] As used herein, the term "antibody" refers generally and broadly to immunoglobulins, autoantibodies, monoclonal antibodies, and polyclonal antibodies, as well as active fragments thereof. The fragment may be active in that it binds to the cognate antigen, or it may be active in that it is biologically functional. The antibodies of the invention may be chimeric, humanized, or human, using techniques known in the art.
[0081] As used herein, the term "monoclonal antibody" refers to antibodies that are chemically and immunologically homogeneous, generally produced by hybridomas. See A Laboratory Manual, Harlow and Lane, eds., Cold Spring Harbor, N.Y. (1988).
[0082] As used herein, the term "polyclonal antibody" refers to antibodies that are produced by more than one clone of antibody-synthesizing plasma cells (B-lymphocytes) in response to the same antigen. They are generally produced by the animal after it is immunized with the antigen.
[0083] As used herein, the term "chimeric antibody" refers to antibodies that contain sequences from more than one source. For example, such antibodies may contain sequences from non-human sources that are then modified by introducing human sequences. As used herein, the term "humanized antibody" refers to an antibody in which minimal portions of a non-human antibody are introduced into an otherwise human antibody. As used herein, the term "human antibody" refers to an antibody in which substantially every part of the protein is substantially non-immunogenic in humans, with only minor sequence changes or variations.
[0084] As used herein, the term "alpha-enolase specific antibody" refers to an antibody that has a high specificity for mammalian ENOl, but not to EN02 or EN03. Similarly, the term "ENOl -specific antibody" refers to an antibody that binds the alpha-enolase protein.
[0085] The term "neutralizing" when referring to a targeted binding agent, such as an antibody, relates to the ability of said targeted binding agent to eliminate, or significantly reduce, the activity of a target antigen. Accordingly, a "neutralizing" ENOl antibody is capable of eliminating or significantly reducing the activity of ENOl. A neutralizing ENOl antibody may, for example, act by blocking the binding of ENOl to the plasminogen. By blocking this binding, the plasminogen mediated cell dissociation is significantly, or completely, eliminated. Ideally, a neutralizing antibody against ENOl enhances cell adhesion.
[0086] The term "polypeptide" is used herein as a generic term to refer to native protein, fragments, or analogs of a polypeptide sequence. Hence, native protein, fragments, and analogs are species of the polypeptide genus. Preferred polypeptides in accordance with the invention comprise the human heavy chain immunoglobulin molecules and the human kappa light chain immunoglobulin molecules, as well as antibody molecules formed by combinations comprising the heavy chain immunoglobulin molecules with light chain immunoglobulin molecules, such as the kappa or lambda light chain immunoglobulin molecules, and vice versa, as well as fragments and analogs thereof. Preferred polypeptides in accordance with the invention may also comprise solely the human heavy chain immunoglobulin molecules or fragments thereof.
[0087] The term "polynucleotide" as referred to herein means a polymeric form of nucleotides of at least 10 bases in length, either ribonucleotides or deoxynucleotides or a modified form of either type of nucleotide, or RNA-DNA hetero-duplexes. The term includes single and double stranded forms of DNA.
[0088] The term "CDR region" or "CDR" is intended to indicate the hypervariable regions of the heavy or light chains of the immunoglobulin as defined by Kabat et al., 1991 ( abat, E. A. et al., (1991) Sequences of Proteins of Immunological Interest, 5th Edition. US Department of Health and Human Services, Public Service, NIH, Washington), and later editions. An antibody typically contains 3 heavy chain CDRs and 3 light chain CDRs. The term CDR or CDRs is used here in order to indicate, according to the case, one of these regions or several, or even the whole, of these regions which contain the majority of the amino acid residues responsible for the binding by affinity of the antibody for the antigen or the epitope which it recognizes.
[0089] Among the six short CDR sequences, the third CDR of the heavy chain
(HCDR3) has a greater size variability (greater diversity essentially due to the mechanisms of arrangement of the genes which give rise to it). It may be as short as 2 amino acids, although the longest size known is 26. CDR length may also vary according to the length that can be accommodated by the particular underlying framework. Functionally, HCDR3 plays a role in part in the determination of the specificity of the antibody (Segal et al, PNAS, 71:4298-4302, 1974).
[0090] The term a "set of CDRs" referred to herein comprises CDR1, CDR2 and
CDR3. Thus, a set of HCDRs refers to HCDR1, HCDR2 and HCDR3 (HCDR refers to a variable heavy chain CDR), and a set of LCDRs refers to LCDR1, LCDR2 and LCDR3 (LCDR refers to a variable light chain CDR). Unless otherwise stated, a "set of CDRs" includes HCDRs and LCDRs.
[0091] The term "corresponds to" is used herein to mean that a polynucleotide sequence is homologous (i.e., is identical, not strictly evolutionarily related) to all or a portion of a reference polynucleotide sequence, or that a polypeptide sequence is identical to a reference polypeptide sequence.
[0092] In contradistinction, the term "complementary to" is used herein to mean that the complementary sequence is homologous to all or a portion of a reference polynucleotide sequence. For illustration, the nucleotide sequence "TATAC" corresponds to a reference sequence "TATAC" and is complementary to a reference sequence "GTATA."
[0093] The term "sequence identity" means that two polynucleotide or amino acid sequences are identical (i.e., on a nucleotide -by-nucleotide or residue-by-residue basis) over the comparison window. The term "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I) or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
[0094] The terms "substantial identity" or "substantially identical" as used herein denotes a characteristic of a polynucleotide or amino acid sequence, wherein the polynucleotide or amino acid comprises a sequence that has at least 85 percent sequence identity, preferably at least 90 to 95 percent sequence identity, more preferably at least 99 percent sequence identity, as compared to a reference sequence over a comparison window of at least 18 nucleotide (6 amino acid) positions, frequently over a window of at least 24-48 nucleotide (8-16 amino acid) positions, wherein the percentage of sequence identity is calculated by comparing the reference sequence to the sequence which may include deletions or additions which total 20 percent or less of the reference sequence over the comparison window. The reference sequence may be a subset of a larger sequence.
[0095] As used herein, the twenty conventional amino acids and their abbreviations follow conventional usage. Stereoisomers (e.g., D-amino acids) of the twenty conventionalamino acids, unnatural amino acids such as ,α-disubstituted amino acids, N-alkyl amino acids, lactic acid, and other unconventional amino acids may also be suitable components for polypeptides of the present invention. Examples of unconventional amino acids include: 4-hydroxyproline, γ-carboxyglutamate, ε-Ν,Ν,Ν-trimethyllysine, ε-Ν-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, σ-Ν-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In the polypeptide notation used herein, the left-hand direction is the amino terminal direction and the right-hand direction is the carboxy-terminal direction, in accordance with standard usage and convention.
[0096] Similarly, unless specified otherwise, the left-hand end of single-stranded polynucleotide sequences is the 5' end; the left-hand direction of double-stranded polynucleotide sequences is referred to as the 5' direction. The direction of 5' to 3' addition of nascent RNA transcripts is referred to as the transcription direction; sequence regions on the DNA strand having the same sequence as the RNA and which are 5' to the 5' end of the RNA transcript are referred to as "upstream sequences"; sequence regions on the DNA strand having the same sequence as the RNA and which are 3' to the 3' end of the RNA transcript are referred to as "downstream sequences".
[0097] As applied to polypeptides, the term "substantial identity" means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 80 percent sequence identity, preferably at least 90 percent sequence identity, more preferably at least 95 percent sequence identity, and most preferably at least 99 percent sequence identity. Preferably, residue positions that are not identical differ by conservative amino acid substitutions.
[0098] As discussed herein, minor variations in the amino acid sequences of antibodies or immunoglobulin molecules are contemplated as being encompassed by the present invention, providing that the variations in the amino acid sequence maintain at least about 75%, more preferably at least 80%, 90%, 95%, and most preferably about 99% sequence identity to the antibodies or immunoglobulin molecules described herein. In particular, conservative amino acid replacements are contemplated.
[0099] Whether an amino acid change results in a functional peptide can readily be determined by assaying the specific activity of the polypeptide derivative. Assays are described in detail herein. Fragments or analogs of antibodies or immunoglobulin molecules can be readily prepared by those of ordinary skill in the art. Preferred amino- and carboxyl-termini of fragments or analogs occur near boundaries of functional domains. Structural and functional domains can be identified by comparison of the nucleotide and/or amino acid sequence data to public or proprietary sequence databases. Methods to identify protein sequences that fold into a known three-dimensional structure are known. Bowie et al., (1991) Science 253:164. Thus, the foregoing examples demonstrate that those of skill in the art can recognize sequence motifs and structural conformations that may be used to define structural and functional domains in accordance with the antibodies described herein.
[00100] A further aspect of the invention is a targeting binding agent or an antibody molecule comprising a VH domain that has at least about 60, 70, 80, 85, 90, 95, 98 or about 99% amino acid sequence identity with a VH domain of any of antibodies shown in sequences 1, the appended sequence listing, an antibody described herein, or with an HCDR (e.g., HCDR1, HCDR2, or HCDR3) shown in sequences 1. The targeting binding agent or antibody molecule may optionally also comprise a VL domain that has at least about 60, 70, 80, 85, 90, 95, 98 or about 99% amino acid sequence identity with a VL domain any of antibodies shown in sequences 2, the appended sequence listing, an antibody described herein, or with an LCDR (e.g., LCDR1, LCDR2, or LCDR3) shown in sequences 2. Algorithms that can be used to calculate % identity of two amino acid sequences comprise e.g. BLAST (Altschul et al., (1990) J. Mol. Biol. 215: 405-410), FASTA (Pearson and Lipman (1988) PNAS USA 85: 2444-2448), or the Smith- Waterman algorithm (Smith and Waterman (1981) J. Mol. Biol. 147: 195-197), e.g. employing default parameters. In some embodiments, the targeting binding agent or antibody that shares amino acid sequence identity as describes above, exhibits substantially the same activity as the antibodies referenced. For instance, substantially the same activity comprises at least one activity that differed from the activity of the references antibodies by no more that about 50%, 40%, 30%, 20%, 10%, 5%, 2%, 1% or less.
[00101] An antigen binding site is generally formed by the variable heavy (VH) and variable light (VL) immunoglobulin domains, with the antigen-binding interface formed by six surface polypeptide loops, termed complimentarity determining regions (CDRs). There are three CDRs in each VH (HCDR1, HCDR2, HCDR3) and in each VL (LCDR1, LCDR2, LCDR3), together with framework regions (FRs). [00102] Typically, a VH domain is paired with a VL domain to provide an antibody antigen-binding site, although a VH or VL domain alone may be used to bind antigen. The VH domain (e.g. from sequence 1) may be paired with the VL domain (e.g. from sequence 2), so that an antibody antigen-binding site is formed comprising both the VH and VL domains. Analogous embodiments are provided for the other VH and VL domains disclosed herein. In other embodiments, VH chains in sequences 1 are paired with a heterologous VL domain. Light-chain promiscuity is well established in the art. Again, analogous embodiments are provided by the invention for the other VH and VL domains disclosed herein. Thus, the VH of the parent or of any of antibodies chain on sequences 2 may be paired with the VL of the parent or of any of antibodies on sequences 1, and 2or other antibody.
[00103] An antigen binding site may comprise a set of H and/or L CDRs of the parent antibody or any of antibodies in sequences 1 and 2with as many as twenty, sixteen, ten, nine or fewer, e.g. one, two, three, four or five, amino acid additions, substitutions, deletions, and/or insertions within the disclosed set of H and/or L CDRs. Such modifications may potentially be made at any residue within the set of CDRs.
[00104] Preferred amino acid substitutions are those which: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter binding affinities, and (4) confer or modify other physicochemical or functional properties of such analogs. Analogs can include various mutant proteins of a sequence other than the naturally-occurring peptide sequence. For example, single or multiple amino acid substitutions (preferably conservative amino acid substitutions) may be made in the naturally-occurring sequence (preferably in the portion of the polypeptide outside the domain(s) forming intermolecular contacts. A conservative amino acid substitution should not substantially change the structural characteristics of the parent sequence (e.g., a replacement amino acid should not tend to break a helix that occurs in the parent sequence, or disrupt other types of secondary structure that characterizes the parent sequence).
[00105] A further aspect of the invention is an antibody molecule comprising a VH domain that has at least about 60, 70, 80, 85, 90, 95, 98 or about 99% amino acid sequence identity with a VH domain of any of antibodies listed in sequence 1, the appended sequence listing or described herein, or with an HCDR (e.g., HCDR1, HCDR2, or HCDR3) shown in sequences 1. The antibody molecule may optionally also comprise a VL domain that has at least 60, 70, 80, 85, 90, 95, 98 or 99% amino acid sequence identity with a VL domain of any of the antibodies shown in sequences 2, the appended sequence listing or described herein, or with an LCDR (e.g., LCDRl, LCDR2, or LCDR3) shown in sequence 2. Algorithms that can be used to calculate % identity of two amino acid sequences comprise e.g. BLAST, FASTA, or the Smith- Waterman algorithm.
[00106] Variants of the VH and VL domains and CDRs of the present invention, including those for which amino acid sequences are set out herein, and which can be employed in targeting agents and antibodies for human ENOl protein can be obtained by means of methods of sequence alteration or mutation and screening for antigen targeting with desired characteristics. Examples of desired characteristics include but are not limited to: increased binding affinity for antigen relative to known antibodies which are specific for the antigen; increased neutralization of an antigen activity relative to known antibodies which are specific for the antigen if the activity is known; specified competitive ability with a known antibody or ligand to the antigen at a specific molar ratio; ability to immunoprecipitate complex; ability to bind to a specified epitope; linear epitope, e.g. peptide sequence identified using peptide-binding scan as described herein, e.g. using peptides screened in linear and/or constrained conformation; conformational epitope, formed by non-continuous residues; ability to modulate a new biological activity of human ENOl protein or downstream molecule. Such methods are also provided herein.
[00107] A further aspect of the present invention relates to a targeted binding agent(i.e. an antibody) including those for which amino acid sequencesthat binds to the epitope peptide comprising amino acid sequence that has at least about 60, 70, 80, 85, 90, or about 92% amino acid sequence identity listed in sequences 9 or lOon human ENOl protein and can be used to treat an human ENOl protein disease or disorder. A human ENOl protein-related disease or disorder can be any condition arising due to the aberrant activation or expression of human ENOl protein. In one example, the human ENOl protein-related disease is a neoplastic disease such as non-small cell lung cancer, hepatocellular (liver) carcinoma, gastric (stomach) cancer, breast cancer, pancreatic duct adenocarcinoma.
[00108] An antibody antigen-binding site composed of a VH domain and a VL domain is typically formed by six loops of polypeptide: three from the light chain variable domain (VL) and three from the heavy chain variable domain (VH). Analysis of antibodies of known atomic structure has elucidated relationships between the sequence and three-dimensional structure of antibody combining sites. These relationships imply that, except for the third region (loop) in VH domains, binding site loops have one of a small number of main-chain conformations: canonical structures. The canonical structure formed in a particular loop has been shown to be determined by its size and the presence of certain residues at key sites in both the loop and in framework regions.
[00109] The techniques required to make substitutions within amino acid sequences of CDRs, antibody VH or VL domains and/or binding agents generally are available in the art. Variant sequences may be made, with substitutions that may or may not be predicted to have a minimal or beneficial effect on activity, and tested for ability to bind and/or neutralize and/or for any other desired property. Variable domain amino acid sequence variants of any of the VH and VL domains whose sequences are specifically disclosed herein may be employed in accordance with the present invention, as discussed.
[00110] The term "polypeptide fragment" as used herein refers to a polypeptide that has an amino-terminal and/or carboxy-terminal deletion, but where the remaining amino acid sequence is identical to the corresponding positions in the naturally-occurring sequence deduced, for example, from a full-length cDNA sequence. Fragments typically are at least about 5, 6, 8 or 10 amino acids long, preferably at least about 14 amino acids long, more preferably at least about 20 amino acids long, usually at least about 50 amino acids long, and even more preferably at least about 70 amino acids long. The term "analog" as used herein refers to polypeptides which are comprised of a segment of at least about 25 amino acids that has substantial identity to a portion of a deduced amino acid sequence and which has at least one of the following properties: (1) specific binding to human ENOl protein under suitable binding conditions, (2) ability to block appropriate ligand/ ENOl protein binding, or (3) ability to inhibit ENOl protein activity. Typically, polypeptide analogs comprise a conservative amino acid substitution (or addition or deletion) with respect to the naturally-occurring sequence. Analogs typically are at least 20 amino acids long, preferably at least 50 amino acids long or longer, and can often be as long as a full-length naturally-occurring polypeptide.
[00111] As used herein, a "targeted binding agent" is an agent, e.g. antibody, or binding fragment thereof, that preferentially binds to a target site. In one embodiment, the targeted binding agent is specific for only one target site. In other embodiments, the targeted binding agent is specific for more than one target site. In one embodiment, the targeted binding agent may be a monoclonal antibody and the target site may be an epitope. As described below, a targeted binding agent may comprise at least one antigen binding domain of an antibody, wherein said domain is fused or contained within a heterologous protein.
[00112] An antibody may be oligoclonal, a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a CDR-grafted antibody, a multi-specific antibody, a bi-specific antibody, a catalytic antibody, a chimeric antibody, a humanized antibody, a fully human antibody, an anti-idiotypic antibody and antibodies that can be labeled in soluble or bound form as well as fragments, variants or derivatives thereof, either alone or in combination with other amino acid sequences provided by known techniques. An antibody may be from any species. The term antibody also includes binding fragments of the antibodies of the invention; exemplary fragments include Fv, Fab, Fab', single stranded antibody (svFC), dimeric variable region (Diabody) and disulfide stabilized variable region (dsFv). "Binding fragments" of an antibody may be produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies. Binding fragments include Fab, Fab', F(ab')2, Fv, and single-chain antibodies.
[00113] Digestion of antibodies with the enzyme, papain, results in two identical antigen-binding fragments, known also as "Fab" fragments, and a "Fc" fragment, having no antigen-binding activity but having the ability to crystallize. Digestion of antibodies with the enzyme, pepsin, results in the a F(ab')2 fragment in which the two arms of the antibody molecule remain linked and comprise two-antigen binding sites. The F(ab')2 fragment has the ability to crosslink antigen. "Fv" when used herein refers to the minimum fragment of an antibody that retains both antigen-recognition and antigen-binding sites. "Fab" when used herein refers to a fragment of an antibody that comprises the constant domain of the light chain and the CHI domain of the heavy chain. The term "mAb" refers to monoclonal antibody.
[00114] The term "epitope" includes any protein determinant capable of specific binding to an immunoglobulin or T-cell receptor. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and may, but not always, have specific three-dimensional structural characteristics, as well as specific charge characteristics. An antibody is said to specifically bind an antigen when the dissociation constant is < 1 micro M, preferably <100 nM, and most preferably < 10 nM.
[00115] "Active" or "activity" in regard to anENOl polypeptide refers to a portion of anENOl polypeptide that has a biological or an immunological activity of a native ENOl polypeptide. "Biological" when used herein refers to a biological function that results from the activity of the native ENOl polypeptide. A preferred ENOl biological activity includes, for example, ENOl induced the plasminogen activity.
[00116] "Mammal" when used herein refers to any animal that is considered a mammal. Preferably, the mammal is human. The term "subject" includes human and veterinary subjects.
[00117] The term "pharmaceutical agent or drug" as used herein refers to a chemical compound or composition capable of inducing a desired therapeutic effect when properly administered to a patient. Other chemistry terms herein are used according to conventional usage in the art, as exemplified by The McGraw-Hill Dictionary of Chemical Terms (Parker, S., Ed., McGraw-Hill, San Francisco (1985)).
[00118] As used herein, "substantially pure" means an object species is the predominant species present (i.e., on a molar basis it is more abundant than any other individual species in the composition), and preferably a substantially purified fraction is a composition wherein the object species comprises at least about 50 percent (on a molar basis) of all macromolecular species present. Generally, a substantially pure composition will comprise more than about 80 percent of all macromolecular species present in the composition, more preferably more than about 85%, 90%, 95%, and 99%. Most preferably, the object species is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species.
[00119] As used herein, the term "monitoring" refers to the process of detecting and/or observing the development of cancer by determining the abundance of ENOl protein in cancer cells.
[00120] Methods for determining the abundance of ENOl include, but are not limited to, measuring the binding of ENOl proteins and ENOl -specific antibodies, Western blotting, flow cytometry, immunohistochemistry (IHC), RT-PCR, and/or microarray analysis.
EXAMPLES
[00121] The practice of the present invention will employ technologies comprising conventional techniques of cell biology, cell culture, antibody technology, and genetic engineering, which are within the ordinary skills of the art. Such techniques are explained fully in the literature.
[00122] The following examples illustrate the development and use of ENOl -specific antibodies to suppress tumor growth by inducing an anti-ENOl immune response.
Example 1
Cell surface location of ENOl in malignant cells
[00123] To analyze the cell location of ENOl on the malignant cells, the flow cytometrywas performed toconfirm the cell-surface localization of ENOl. Tumor cells were obtained from pleural effusions of 17 patients with lung cancer. Among them, tumor cells obtained from 2 cancer patients, NHRI-L89 and CA926 effusion tumor cells, are representative cases shown here. NHBE and SAEC cells are normal human lung primary cells. Intact whole cells were stained with or without ENOl antiserum and analyzed for the surface ENOl distribution using the antibody specific to ENOl protein in flow cytometric analysis (as shown in FIG. lA).The cell localization of ENOl of lung cancer cell line NHRI-L89is further confirmed by immunohistology (FIG. IB). Briefly, lxlO4 of NHRI-L89 cells were grown in DMEM containing 10% FCS on a chamber slide coated with fibronectin. After overnight culture, the slide was washed withPBS and blocked with goat serum for 1 hour. NHRI-L89 intact whole cells were stained with control (CTL) or ENOl antibody for surface ENOl, followed by PI staining for nuclear detection. The stainings were visualized under a confocal microscope (400x; FIG. IB). [00124] The results of this study areshown in FIG. 1A (flow cytometry) and IB (immunostaining).Incubating NHRI-L89 and CA926 with ENOl antibody shifts the histogram to the right, as compared to incubating the cells with control antibody .Normal NHBE and SAEC human lung primary cells show no significant shifts (FIG. 1 A). This result indicates that the ENOl protein is expressedon the lung cancer cells, but not on normal primary cells ( FIG. 1 A). When the cell localization of ENOl wasfurther analyzed by immunohistology,intact NHRI-L89cells stained with ENOl antibody shows the signals on the cell surface (FIG. IB). This result suggests that ENOl is expressed on the surface of lung and some cancer cells and, therefore, can be a potential target for immune therapy.
Example 2
Anti-ENOl antibody inhibits the invasion capacity of human lung cancer Cells
[00125] To investigate the effects ofENOl antibody on cancer cell invasion, the invasion activity of the CLl-5 cells was assessed using a Transwell assay by addingENOl antibodyto CLl-5 cells grown on micropore filters (Becton Dickinson, Franklin Lakes, N.J.) coated with extracellular matrix (matrigel) (Becton Dickinson). After being mixed with 1 :20, 1:100, and 1:500 dilutions of anti-ENOl polyclonal antibody, respectively, 2xl04 cells of CLl-5 were seeded in the top chamber with media containing 2% FBS of a two-chamber assay system and incubated for 24 hours with 10% FBS medium in the lower chamber. An anti-GST mouse polyclonal antibody was used as a negative control group. Two chambers were separated by a micropore filter (12 micrometer pore size) coated with matrigel. After the incubation period, the matrigel-coated filters were stained and the number of cells invading into the matrigel-coated filter was quantified under a microscope. The study was repeated three times. Data are presented as mean± SD. Thetest was used to compare activity between each group. The P values <0.05 were considered statistically significant.
[00126] The resultsare shown in the FIG. 2. There are no significant cell count difference between the non-treated (160±8; N=3) and the anti-GST polyclonal treated group(168±12; N=3). However, when CLl-5 cells were administrated with different dilutions of ENOl polyclonal antibody, cell counts of CLl-5 are reduced, rangingfrom 52±12(N = 3) for the 1 :20 dilutionto 156±30 (N=3)for the 1:500 dilution; the reduction is proportional to the concentrations of ENOl polyclonal antibody. There is statisticscell count difference between the 1:20 dilution of ENOl polyclonal and 1:20 dilution of anti-GST polyclonalgroups (P<0.05). Theseresults suggest that the administration of ENOl antibody to CLl-5 cancer cells inhibits the cell invasion activity in vitro.
Example 3
ENOl polyclonal antibody attenuates the tissue invasion capability of CL1-5F4 cells in lung. [00127] To investigatethe effect of administration of ENOl antibody on metastasis of lung cancer cells and evaluate the potential of anti ENOl antibody in cancer therapy, highly-invasive CL1-5F4 cells with a luciferase reporter gene (l xlO6 cells/mouse) were intravenously injected into NOD-SCID mice,together with an ENOl antibody or a control (CTL) antiserum twice per week. The lung metastatic colony formation was monitored 12 and 19 days after tumor injection using an IVIS system. The mouse injected with CL1-5F4 cells with knockdown of ENOl gene was used as a negative control. After the mice were scarified ondays 12 and 19, the metastatic colonies and tumor sizes were visualized and counted.
[00128] The results are shown in FIG.3. When a mouse bearing CL1-5F4 cells with a luciferase reporter gene was administrated with a control antibody, the lung of mouse started to show fluorescence signals on day 12. On the other hand,signals were not detected in the mouse treated with ENOl polyclonal antibody or inthe mouse bearing CL1-5F4 cells and having knockdown of ENOl gene. On the day 19, the signal of mouse treated with control antibodyappearedstronger, indicatingthe presence of more cell invasion or tumor growth. On the same day,signals were stillnot detected in the mouse treated with ENOl polyclonal antibody or inthe mouse bearing CL1-5F4 cells with knockdown of ENOl gene. These results indicate that the metastasis of CL1-5F4 cells was compromised by ENOl antibody orENOl gene knock-down (FIG.3; Top panel).After days 12 and 19, lungs of each treatment group were removed and tumor nodules were determined. The resultsare shown in the bottompanels in FIG.3. There are a few tumor nodules appearing in the control anti-body treatment mouse with clear tumor sizes. However, the mouse treated with ENOl polyclonal antibody ormouse bearing CL1-5F4EN01 knockdown showed no clear tumor nodules, indicating the delay of tumor metastasis in these two groupsof mice. The results of this study suggest thatadministrating ENOl antibody has anti-metastasis or anti-proliferation effects on cancer cells in vivo and ENOl antibody has potential applications in cancer therapy.
Example 4
The generation of anti-human ENOl monoclonal antibody.
[00129] From examples 2 and 3, it is clear that ENOl antibodycan attenuate the invasion and metastasis of cancer cells. Therefore, ENOl antibody can potentially be developedas a therapeutic antibody. To generate a monoclonal antibody against human ENOl, BALB/c mice were primed with purified recombinant human ENOl antigen (50 μg/mouse), followed by boosting the ENOl humoral responses with an emulsified CpGadjuvant. The splenocytes were harvested, fused with Fo cells, and then diluted according to a standard protocol. The hybridoma cells secreting monoclonal antibodies that recognize the ENOl antigen were selected by an antigen-based ELISA. The selected clones were then verified by the binding activity of the antibodies to the cell-surface ENOl on CL1-5F4 lung adenocarcinoma cells, using intact whole cell staining in flow cytometric analysis, as described in Example 1.
[00130] The resultsare shownin the FIG.4. All of five antibody clones isolated recognize the cell surface ENOl onCLl-5F4, as evidenced by the histograms shifting to right, as compared with the control antibody.
Example 5
The ENOl binding ELISA of 5 antibody clones isolated
[00131] To investigate the ENOl binding affinities of 5 antibody clones isolated above, the individual hybridomas were grown in RPMI containing 10% FCS. After one week culture lxlO6 cells were collected, washed withPBS,resuspended in 200ul RPMI medium, and injected to a SCID mouse by IP injection. After 3 weeks, acities of mouse was collected and diluted to 15 ml. Antibody was further purified by 40 % ammonium sulfate precipitation and a Protein A column (Montage antibody purification kit Millipore). The purified antibody was concentrated with anAmicon Ultra- 15 centrifugal filterdevice,following the protocols provided by manufacturer (Millpore). The purities of the antibodies were analyzed by 12% SDS PAGE. Human ENOl protein (400ng) was coated on a 96-well ELISA plate and the
1 1 7 plate waswashedwith PBS twice. A series of dilutions from 1 10" tolxlO" M of individual antibodies were added to the plate, and the plate was incubated at 37°C for 1 hour. A goat anti mouse IgG conjugated with HPRT was added and after 1 hour incubation, and thenTMB was added. The OD405 was read to calculate the activity.Every study was repeated three times, andthe data were presented as mean±SD. OD readings and concentrations of antibodies were used to make a multiple scatter plot using Sigmaplot™. The ¾ values of clones were predicted by four parameter logistic fit.
[00132] The results of this experiment are shown in FIG.5.A11 of antibody hybridomas have productivities from 20.4 mg to 4.6mg per mice. TheKd values of the antibodiesrangefrom 1.77xl0"10±0.12 tol.6xl0"5±0.2 M (N=3). These results suggest that these 5 antibody clones recognize the human ENOl protein and thatclone 10 has the best affinity with aKd of about 1.77xlO"10±0.12M (N=3).
Example 6
The plasminogen receptor antagonist activitiesof 5 antibody clones isolated
[00133] To studytheENOl antagonist activities of an isolated anti-human ENOl antibody, a U937human lymphoma cell line was grown in RPMI containing 10%> FCS. Cells were treated withlO microgram/ml of LPS for 6 hours to induce ENOl protein expression on the cell surface. 1.5xl06cells/ml in PBS were pre-incubated with 1 micro gram/mlof human Lys-plasminogen and 10 microgram/ml of different clonesof anti ENOl antibodies for one hour, respectively. Samples were washed with PBS twice,and then 3nM of tissue specific plasminogen activator and 0.5mM of chromogenic substrate S-2251 were added. After one hour incubation at 37°C, the OD405 was read to calculate the activities. Each study was repeated three times, and the antagonist activities were analyzed. Dataare presented as mean±SD.T-test was used to compare activity between each group. P values <0.05 were considered statistically significant.
[00134] Results of this study are shown in FIG.6.A11 of 5 clone antibodies have plasminogen receptor antagonist activities with inhibition, ranging from 52 % to 100% of LSP-induced ENOl specificactivity. Thesestudies suggest that the plasminogen receptor antagonist activity isproportional to theKdvalue of each clone, and that clone 10 has the best inhibitionactivity,close to 100%inhibition of ENOl specific activityinduced by LPS.
Example 7
ENOl monoclonal antibodyinhibitstheinvasion capacity of human lung Cancer Cells
[00135] The inhibition of the invasion capacity of CLl-5 cellsby each antibody clone was assessed using a Transwell assay using micropore filters (Becton Dickinson, Franklin Lakes, N.J.) coated with extracellular matrix (matrigel) (Becton Dickinson). After mixing with 10 microgram/ml each of 5 clone antibodies, 2*104 cells were seeded in the top chamberof a two-chamber assay system and incubated for 24 hours with media containing 10% FBS in the lower chamber. An anti-mouse IgG and 5-aza-2-deoxycytidine (5 ADC) were used as a negativecontrol and a positive control, respectively. Two chambers were separated by a micropore filter (12micron pore size) coated with matrigel. After the incubation period, the matrigel-coated filters were stained and the number of cells invading into the matrigel-coated filter was quantified under a microscope. Each study was repeated three times. Data were presented as mean± SD. T-test was used to compare activity between each group.P values <0.05 were considered statistically significant.
[00136] Results from these experiments are shown in FIG.7.These results show that all anti-human ENOl antibody clones have inhibitory activitiesagainst the invasioncapacity of CLl-5, and thatclone 10 has the bestinhibition activity, about 65.5+0.3% (N=3) of the control IgG group.
[00137] Taking together the results from Examples 5,6, and 7, anti-human ENOl antibody clones 10 was chosen for further development and referred toas EN 10 mAb in this description.
Example 8
EN 10 mAb inhibitsthe Invasion Capacity of Human Lung Cancer Cells and Lymphoma cells
[00138] The inhibition of the invasion capacity of CLl-5 cellsby EN 10 mAbwas assessed using a Transwell assay using micropore filters (Becton Dickinson, Franklin Lakes, NJ) coated with extracellular matrix (matrigel) (Becton Dickinson). After mixing with 10, 50, and 100 microgram/ml of EN 10 mAb, respectively,2 l04 cellswere seeded in the top chamber with media containing 2% FBS in a two-chamber assay system and incubated for 24 hours with 10% FBS medium in the lower chamber. An anti-mouse IgG was used as a negative control. Two chambers were separated by a micropore filter (12μηι pore size) coated with matrigel. After the incubation period, the matrigel-coated filters were stained, and the number of cells invading into the matrigel-coated filter was quantified under a microscope. Each study was repeated three times. Data are presented as mean±SD. T-test was used to compare activities between each group. P values <0.05 are considered statistically significant. The resultsareshown in Fig.8 A.
[00139] Similarly, inhibition of the invasion capacity of U937 cellsby EN10 mAbwas assessed using a Transwell assay using micropore filters (Becton Dickinson, Franklin Lakes, NJ) coated with extracellular matrix (matrigel) (Becton Dickinson). A human lymphoma U937 cell line was grown in RPMI containing 10% FCS. Cells were treated with 10 microgram/ml of LPS for 6 hours to induce ENOl protein expression on cell surface. After mixing with 10, 50, and 100 microgram/ml of EN 10 mAb, respectively, 2 l04 cells were seeded in the top chamber of a two-chamber assay system and incubated for 24 hours with media containing 10% FBS and lOnM MCP-1 in the lower chamber. An anti-mouse IgG was used as a negative control. Two chambers were separated by a micropore filter (8μπι pore size) coated with matrigel. After the incubation period, cell in the lower chamber were counted by a hemocytometer under a microscope. Each study was repeated three times. Data were presented as mean±SD. T-test was used to compare activity between each group. P values <0.05 are considered statistically significant.
[00140] The resultsare shown in the FIG. 8A and 8B.Theinvasion activity of CLl-5 was inhibited to an extent ranging from 31+2 to 77±1 % (N=3) of the control IgG, when cells were administrated with EN10 mAb from 10 microgram/ml to 100 microgram/ml ( FIG. 8A). These resultsare similar to thoseof Example 4 and the inhibition is proportional to the concentrations of EN 10 mAb with anEC5o estimated to be about 3 Omicro gram/ml.
[00141] To investigatewhether theinhibition of invasion is applicable to the other cancer cells, U937 cells were treated with EN10 mAb ranging from 5 microgram/ml to 50 microgram/ml in a similar manner. The invasion activity of U937 was inhibited to an extent ranging from 91.2±2 to 49.1±1% (N=3) of the control IgG (FIG. 8B). Theseresults indicatethat invasion activities of CLl-5 andU937 are inhibited byENIO mAbinadose-dependent manner, presumably by inhibitingthe plasminogen receptor activity of ENOl.
Example 9
EN10 mAb recognizes LSP-Induced surfaceENOl in lymphoma Cells [00142] A human lymphoma U937 cell line was grown in RPMI containing 10% FCS. Cells were treated with 1 microgram/ml of LPS for 6 hours to induce ENOl protein expression on the cell surface.For flow cytometric analysis, the intact whole cells were stained with or without EN10 mAb (1 :300 dilution), visualized with FITC-conjugated goat anti mouse IgG (Jackson Lab), and analyzed with FACScan flow cytometer (Becton Dickinson). ENOl expression is measured by the resulting fluorescence intensity.
[00143] Results from these experiments are shown in FIG. 9. Incubating U937 with LPS and treatment withENlO mAb shifts the histogram to the right, as compared to incubating the cells without LPS treatment butwith EN 10 mAb treatment, indicating that U937 cells express ENOl on their cell surfaces. These data support that EN10 mAb recognizes the LSP- Induced surfaceENOl on lymphoma cells
Example 10
EN10 mAb inhibits the dissociation of CL-5 cells from collagen and fibronectin
[00144] To assess the signal transduction pathway betweenENOl plasminogen receptor-plasminand extracellular substrates, lmg/ml of gelatin, 100 microgram/ml of fibrinogen, 10 microgram/ml of collagen and 10 microgram/ml of fibronectin, respectively, were coated on a non-treated ELISA plate overnight. CLl-5 cells (4><104cells) were seeded on the plate, and 50 microgram/ml of EN10 mAb was added to 200μί of DMEM containing 10% FCS. Cells were incubated at 37°c for 24 hours and then washed with PBS twice. 10%) WST was added andreaction mixtures were incubated at 37°C for 4 hours. The relative cell numbers in the plate were estimated by the reading of OD450. Each study was repeated three times. Data are presented as mean±SD. T-test was used to compare activity between each group. P values <0.05 are considered statistically significant.
[00145] Results from these experiments are shown in FIG.1 OA. Thesedata indicate that OD450 readings on fibronectin and collagen coated plates are 2.45+0.37(N=3)and 1.83± 0.44 (N=3).The readings aremuch higher than those of gelatin and fibrinogen plates, which are not significant different from the background reading. There are no significant difference between the EN 10 mAb treated group and the non-treated group. Theseresults suggest that CLl-5 cells favor binding to fibronectin and collagen, and EN 10 mAbisnot involved in the cell association pathway when cells are incubated in the medium without down-stream proteases,for example plasminogen and tPA.Data in FIG. 10A suggest that the ENOl plasminogen receptor activity isnot involved in the cell association pathway in the extracellular matrix.
[00146] We further testedwhether ENOl takes part in the cell dissociation pathway in the extracellular matrix. One microgram/ml of fibronectin and 10 microgram/ml of collagen were, respectively,coated on a non-treated ELISA plate overnig ht.CLl-5 cells (4xl04cells) were seeded on the plate, and 0, 6.25, 12.5, 25, and 50 microgram/ml of EN10 mAb, respectively, were added to 200 micro L of DMEM containing 10% FCS.Furthermore,10 microgram/mL Glu-plasminogen and 2nM tPA were added. Cells were incubated at 37°C for 24 hours and washed with PBS twice. Then, 10% WST was added and reaction mixtures were incubated at 37°C for 4 hours. The relative cell numbers in the plate were estimated by the readings of OD450. Each study was repeated three times.Data are presented as mean±SD. T-test was used to compare activity between each group. P values <0.05 are considered statistically significant.
[00147] Results from these experiments are shown in FIG.10B and FIG. IOC. The data indicate that cell numbers are directly proportional to the concentrations of treated EN10 mAbin both extracellular matrices when the medium contains the ENOl receptor down-stream proteases plasminogen and tPA.There are significant difference between 50 microgram EN10 mAb treated group andthe control IgG group (P<0.05) in both extracellular matrix studies. These results suggest that ENOl is involved in the dissociation pathway of CLl-5 cells from extracellular matrixes, presumably by enhancing the plasmin and tPA protease activity.ENIO mAb, functioningas an antagonist of ENOl, blocks the receptor activity of ENOl, resulting in the inhibition ofplasmin and tPA activation and, therefore, inhibits the dissociation activity of CLl-5 cells from extracellular matrixes and invasion.
Example 11
Preparation of Genes Encoding Monoclonal Antibodies
[00148] Cloning of the gene encoding the antibodyENIO mAb is performedin accordance with the methods described below.
(1) cDNA Cloning of Antibody Genes and Preparation
[00149] The hybridoma was cultured in a RPMI medium (manufactured by Gibco) containing 10% FCS. After the cell number reached about 10> 106/ml, the cells were collected by centrifugation, and then TRIzol® (manufactured by Invitrogen) was added to extract total RNA in accordance the instruction manual. Cloning f the variable region of the antibody cDNAs was performed using a mouse Ig-primer set (manufactured byNovagen) in accordance with the attached instruction manual.
(a) The synthesis of 1st Strand cDNA was performed in accordance with the instruction manual of Superscript® III First-Strand Synthesis System (manufactured by Invitrogen).
[00150] The 1st strand cDNA was prepared using 5microgram of the total RNA as a template. Five microgram of total hybridoma RNA, 1. microL of 50ng/microL of random primers, and ImicroL of lOmM dNTP were mixed, and DEPC-treated water was added to lOmicroL in a 200 1 PCR tube. The reaction mixture was incubated at 65 °C for 5 min, and then placed on ice for at least 1 minute. Ten (10) microLof cDNA Synthesis Mixture containing 2microL of 10x RT buffer, 4 microL of 25mM MgCl2, 2microL of DTT, ImicroL of 4 unitRNaseOUT™, and ImicroL of 200 unit Superscript® III RT were added, mixed gently, and collected by brief centrifugation. The reaction tube wasincubated for 10 min at 25°C and followed by50 min at 50°C. The reaction was terminated at 85°C for 5 min and chilled on ice.The tube was briefly centrifuged to collect the reaction, and 1 microL of RNase H was added and incubated for 20 min at 37°C.
(b) Amplification by PCR of heavy chain genes and light Chain genes
[00151] A reaction solution having a composition of 5 microLof cDNA, 5 microL of 10x reaction Buffer, 1 microL of lOmM dNTP mix, ImicroLof 2.5 unit Taq polymerase, and ImicroL of forward primer 1 andl microL of reverse primer 2 provided bythe primer set was prepared in a final volume of 50 microL with double distilled water and subjected to PCR.
[00152] For amplification of the light chain and heavy chain of an antibody, a cycle of 94degree C for 10 minutes was used, then a cycle of 94degreeC for one minute, 52degree C for one minute, and 72degree C for 1 minutewas repeated 35 times, and the reaction was incubated at 72degreeC for 10 moreminutes.The reaction solution was subjected to 2% agarose gel electrophoresis to analyze the reaction products. Products with the correctmolecular weights, about 463 bps for the heavy chain and 451bps for the light chain, were ligated to a pCR 2.1-TOPO vector (manufactured by Invitrogen) for subcloning in accordance with the attached instruction manual. M13forward (5'-GTAAACAAC GACGGCGAG-3 '(SEQ ID NO: l 1) and Ml 3 reverse (5*-CAG GAA ACA GCT ATG AC--3' (SEQ ID NO: 12) ) primers were then used to determine the nucleotide sequence. Based on the sequence information, antibody sequenceswere translated into proteins sequences by ExPASY-Translation Tool. Resulting sequences of EN 10 mAbcomprisea heavy chain amino acid sequence and a light chain sequence having complementarity determining regions (CDR),which were determined by the method published by Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda Md. (1991), vols. 1-3.
[00153] FIG. HAdepicts the variable heavy chain region amino acid sequence of EN10 niAb (SEQ ID NO: 1). The framework regions (FR1, FR2, FR3, and FR4) and CDRs (HCDR1(SEQ ID NO: 3), HCDR2(SEQ ID NO: 4), and HCDR3(SEQ ID NO: 5) are indicated.
[00154] FIG. 11B depicts the variable light chain region amino acid sequences of EN10 mAb (SEQ ID NO: 2). The framework regions (FR1, FR2, FR3, and FR4) and CDRs (LCDR1(SEQ ID NO: 6), LCDR2(SEQ ID NO: 7), and LCDR3(SEQ ID NO: 8) are indicated.
Example 12 Epitope mapping
Antibody Epitope Mapping
[00155] To determine the epitope of ENlOMab on the human ENOl protein, twoforward primers,with thenucleotide sequencesof5 ' -GGATCCGC AGC AAACTTC AGGGAAGCC ATG-3 ' (SEQ ID NO:13), and 5 ' -GGATCCTCGAAGATCCCTTTGACC AGGATG-3 ' ( SEQ ID NO: 14),and a reverse primer(5 ' -TC AGGCTGAAAATCTCTC ATCCGC-3 (SEQ ID NO:15)were designed. AnE.co/z'expression plasmidpTRC-HIS ENOl containing human ENOl cDNA gene was used as atemplate to amplify ENOl deletion mutants. Primers with SEQ NO:13 and SEQ NO:14 were used as forward primers,with the SEQ ID NO: 15 as a reverse primer,to amplifydeletion mutants Al-189(Fig. 12A) and Δ1-297 (Fig. 12A), respectively. The other set of primers, having the sequencesof5 ' -GGATCCTATCTATTCTC AAGATCC ATGCC-3 ' (SEQ ID NO: 16) and 5 ' -CTCGAGGTCATGGTGTCTC ATCGTTCGCTCGAG-3 ' (SEQ ID NO: 17), was used to amplify adeletion Δ297-434 mutant.For amplification of each mutant, a reaction solution having a composition of ImicroL of 1 : 1000 dilution of template DNA about O.lng, 5 microL of 10* reaction buffer, ImicroL of lOmM dNTP mix, 1 microL of 2.5 unit Ta polymerase , ImicroL of the forward primer, andlmicroLof the reverse primer was prepared in a final volume of 50 microLwith double distilled water and subjected to PCR. A cycle of 94degree C for 10 minutes was used, then a cycle of 94degree C for one minute, 52degree C for one minute, and 72degree C for 1 minute was repeated 35 times, and the reaction was incubated at 72degree C for 10 more minutes. This reaction solution was subjected to 2% agarose gel electrophoresis to analyze the reaction products. Reaction products with the correct molecular weights were ligated into a pCR 2.1-TOPO vector (manufactured by Invitrogen) for subcloning in accordance with the attached instruction manual. M13 forward (5'-GTAAACAACGACGGCGAG~3'(SEQ ID NO: 11) and M13 reverse (5'- CAG GAAACA GCT ATG AC--3* (SEQ ID NO: 12)) primers were then used to determine the nucleotide sequence. Every mutation clone with the correctsequences was digested with restriction enzymes BamRl andA¾oI,and the digestion products weresubjected to 2% agarose gel electrophoresis. The insertion fragment of each mutant was cut from the agarose gel and purified with aGene Clean Kit in accordance with the attached instruction manual provided by the manufacturer(BIOlOl). The BamRl and J¾oIDNA fragment of each mutant was ligated to theBamRl and Xhol sites of mE.coli expression vectorpTRC His A (Invitrogen). The resulting plasmid was transformed into E.co/ BL21 Rosseta. The ENOl mutation protein was expressed in E.coli by IPTG induction and purified by Ni-agarose in accordance with the attached instruction manual provided by the manufacturer (Qiagen) after the bacteria pellet was sonicated. The purity of each mutant was analyzed by 12% SDS PAGE.To determine the binding activity of each mutant protein, 400ng of human ENOl protein was coated on a 96-well ELISA plate and plate was washed by PBS. 10 microgram of EN10 mAb was added to the plate and the plate was incubated at 37°C for 1 hours. After the binding complex was washed with PBS twice, agoat anti-mouse IgG conjugated with HPRT was added. After 1 hour incubation, TMB was added. The binding affinity was determined by the readings of OD 405. Each study was repeated three times. Data are presented as mean±SD. T-test was used to compare activity between each group. P values <0.05 were considered statistically significant.
[00156] The results are shown in FIG. 12A.EN01 mutants Al-189and Δ1-297 have OD405 readings about 1.4310.18 and 1.56±0.08 (N=3)(these are 1.43 and 1.56 in FIG. 12A), which areabout 42% and 39% of that of the wild type ENOl (2.87+0.08) (N=3), respectively. However, when amino acid residues from 297to 434 were deleted, the binding activity of this mutated ENOl to EN 10 mAb was lost, as compared with the BSA background. Theseresults suggest that amino acid residues from 297 to 434 are required for ENOl protein bindingto EN10 mAb and the decrease in the binding activity of mutantsAl-182 and Δ1-297 may be due to the instability or conformation change of the mutant proteins.
[00157] To further explore the epitope of EN10 mAbin the ENOl protein,5reverse primers, having sequences of 5'-CTCGAGAGGGATCTTCGATAGACACCACTGGG -3' (SEQ ID NO:18), 5 ' -CTCGAGCTACCTGG ATTCCTGC ACTGGCTG-3 ' (SEQ ID NO:19),5'-CTCGAGACTTCTCGTTCACGGCCTTGGCGATC-3'(SEQ ID NO:20), 5 ' -CTCGAGACTTCTCGTTC ACGGCCTTGGCGATCC-3 ' (SEQ ID NO:21), 5'-CTCGAGCAGTCTCCCCCGAACGATGAGACACC-3' (SEQ ID NO:22), and 5'- CTCGAG CACCAGTCTTGATCTGCCCAGTGC AC-3 ' (SEQ ID NO:23)were designed. AriE.coli expression plasmidpTRC-HIS ENOl containing human ENOl cDNA gene was used as a template to amplify the ENOl deletion mutants. SEQ ID NO: 16 were used as the forward primer to amplify deletion mutants 296-434, 316-434, 336-434, 376-434 and 396-434 with the SEQ ID NO:18, SEQ ID:19, SEQ ID:20, SEQ ID:2l, SEQ ID:22, and SEQ ID:23 primers, respectively. For amplification of each mutant, a reaction solution having a composition of ImicroL of 1 :1000 dilution of template DNA about O.lng, 5 microL of 10x reaction buffer, 1 microL of lOmM dNTP mix, 1 microLof 2.5 unit Taq polymerase, ImicroL of forward primer, and 1 microLof reverse primer was prepared in a final volume of 50 microL with double distilled water and subjected to PCR. A cycle of 94degree C for 10 minutes was used. Then, a cycle of 94degreeC for one minute, 52degreeC for one minute, and 72 degree C for 1 minute was repeated 35 times, and the reaction was incubated at 72degreeC for 10 more minutes. This reaction solution was subjected to 2%> agarose gel electrophoresis to analyze the reaction products. Reaction products with the correct molecular weights were ligated into a pCR 2.1-TOPO vector (manufactured by Invitrogen) for subcloning in accordance with the attached instruction manual. Ml 3 forward (5'-GTAAACAACGACGGCGAG~3'(SEQ ID NO: 11) and M13 reverse (5'-CAGGAA AC AGCT ATG AC--3 ' (SEQ ID NO: 12) primers were then used to determine the nucleotide sequence. Every mutation clone with the correct sequence was digested with restriction enzymes BamHl and Xhol and the digestion product was subjected to 2% agarose gel electrophoresis. The DNA fragment of each mutant was isolated from the agarose gel and purified with a Gene Clean Kit in accordance with the attached instruction manual provided by the manufacturer (BIO 101). The BamHl and Xhol DNA fragment of each mutant was ligatedinto theBamHl and Xhol sites of anE.c /z'expression vector pTRC His A (Invitrogen). The resulting plasmid was transformed into E.coliB ll Rosseta. The ENOl mutation protein was expressed in E.coli y IPTG induction and purified by Ni-agarose in accordance with the attached instruction manual provided by the manufacturer (Qiagen) after the bacteria pellet was sonicated. The purity of each mutant was analyzed by 12% SDS PAGE. To determine the binding activity of each mutant protein, 400ng of human ENOl protein or mutant protein was coated on a 96-well ELISA plate and plate was washed by PBS. 10 microgram of EN 10 mAb was added and incubated at 37°C for 1 hours. After the binding complex was washed with PBS twice, agoat anti-mouse IgG conjugated with HPRT was added.After 1 hour incubation, TMB was added. The binding affinity was determined by the readings of OD 405. Each study was repeated three times. Data are presented as mean±SD. T-test was used to compare activity between each group. P values <0.05 are considered statistically significant.
[00158] The 12% of SDS PAGE of each mutant and the wild type protein areshown in the FIG.12B.The molecular weight of each mutant increases from the mutant 296-343 to the wild type. This result suggeststhat we can yield the whole protein from each mutant even though some degradation of mutants 336-434 and 376-434 can be seen.As showed in the FIG.12C, there is no significant difference between the EN 10 mAb binding affinity of the wild type ENOl and those of deletion mutants336-434, 376-434, and 369-343. However, when amino acid residues from 296 to 316 and 317 to 336 are deleted, the EN 10 mAbbinding activities of these twoENOl mutants arelost,as compared with that theE. coli cell lysatebackground. These results suggest that amino acid residues from 296to 336(FDQDDWGAWQKFTASAGIQVVGDDLTVTNPKR1AKAVNEKS, SEQ ID N0.49)areimportant for ENOl protein binding withENIO mAb.
Example 13 Alanine Scanning
[00159] To further explore which residues from 296 to 336of human ENOl are important for EN 10 mAb binding,the crystal structure of ENOl was downloaded from protein data bank (pdb-entry: 2PSN). After the structure analysis, amino acid residues D300, W301, G302, Q305, K306, A309, K326, K330, N333, E334, and K335 arepredicted to be exposedonthe protein surface and are candidatesfor mutations to analyze whether they are indeed importantfor EN10 mAb binding. 10 of these 11 residues were chosen to be mutated to alanine, except for A309 which was mutated to glycineusingthe QuickChange II site-directed mutagenesis Kitin accordance with the attached instruction manual provided by the manufacturer (Agilent Technology). The following mutagenic oligonucleotides foralaninescanning(Table 1) were generated by Genomics BioScience and Technology Co., Ltd.
TABLEl:01igo
Sequences5 ' -GATCCCTTTGACCAGGATGCCTGGGGAGCTTGGCAG-3 ' (SEQ ID
NO: 24) 5 ' -CTGCCAAGCTCCCCAGGCATCCTGGTCAAAGGGATC-3 ' (SEQ ID NO: 25) 5 ' -CCCTTTGACCAGGATGACGCGGGAGCTTGGCAGAAG-3 ' (SEQ ID
NO: 26) 5' -CTTCTGCCAAGCTCCCGCGTCATCCTGGTCAAAGGG-3' (SEQ ID NO: 27 ) 5 ' -CTTTGACCAGGATGACTGGGCAGCTTGGCAGAAGTTC-3 ' (SEQ ID
NO: 28) 5 ' -GAACTTCTGCCAAGCTGCCCAGTCATCCTGGTCAAAG-3 ' (SEQ
ID NO:29)
5 ' -GACTGGGGAGCTTGGGCGAAGTTCACAGCCAGTGCA-3 ' (SEQ ID NO: 30)
5 ' -TGCACTGGCTGTGAACTTCGCCCAAGCTCCCCAGTC-3 ' (SEQ ID NO: 31) 5' -GGGGAGCTTGGCAGGCGTTCACAGCCAGTGCAGG-3' (SEQ ID NO: 32) 5 ' -CCTGCACTGGCTGTGAACGCCTGCCAAGCTCCCC-3 ' (SEQ ID NO: 33) 5 ' -GGCAGAAGTTCACAGGCAGTGCAGGAATCCAGGTAG-3 ' (SEQ ID NO: 34) 5' -CTACCTGGATTCCTGCACTGCCTGTGAACTTCTGCC-3 ' (SEQ ID NO:35) C5 ' -TCACAGTGACCAACCCAGCGAGGATCGCCAAGGCC-3 ' (SEQ ID NO: 36) 5 ' -GCCTTGGCGATCCTCGCTGGGTTGGTCACTGTGAG-3 ' (SEQ ID NO: 37) 5' -CAACCCAAAGAGGATCGCCGCGGCCGTGAACGAGAAG-3 ' (SEQ ID 0:38) 5 ' -CTTCTCGTTCACGGCCGCGGCGATCCTCTTTGGGTTG-3 ' (SEQ ID NO: 39) 5 ' -GAGGATCGCCAAGGCCGTGGCCGAGAAGTCCTGCAAC-3 ' ( SEQ ID O:40) 5' -GTTGCAGGACTTCTCGGCCACGGCCTTGGCGATCCTC-3 ' (SEQ ID NO: 41) 5' -GATCGCCAAGGCCGTGAACGCGAAGTCCTGCAACTG-3' C(SEQ ID NO: 42) 5' -GCAGTTGCAGGACTTCGCGTTCACGGCCTTGGCGATC-3 ' (SEQ ID NO: 43) 5 ' -GCCAAGGCCGTGAACGAGGCGTCCTGCAACTGCCTC-3 ' (SEQ ID
NO: 44) 5' -GAGGCAGTTGCAGGACGCCTCGTTCACGGCCTTGGC-3 ' (SEQ ID NO: 45)
5' -CAAGGCCGTGAACGCGGCGTCCTGCAACTGCCTCCTG-3 ' (SEQ ID NO: 46) 5' -CAGGAGGCAGTTGCAGGACGCCGCGTTCACGGCCTTG-3' (SEQ ID NO:47)
[00160] For amplification of each mutant, a reaction solution having a composition of 3 microL of template DNA about 30ng, 5 microL of 10x reaction buffer, 1 microL of lOmM dNTP mix, 1 microL of 2.5 unit pfu polymerase, 12.5 microL ofl25ng forward primer, and 12.5 microL of 125ng reverse primer was prepared in a final volume of 50 microL with double distilled water and subjected to PCR. A cycle of 95degreeCfor 10 minutes was used. Then, a cycle of 95degreeC for 30 seconds, 55degreeC for 30 seconds, and 68 degree C for 6 minute was repeated 16 times. After the PCR reaction, 1 microLof Dpnl was added to each PCR tubes, incubated at 37°0 for 1 hour and then Dpnl was heated to be inactivated at 80°C for 20minutes. The reaction products were transformed to 50 microL XL 1 -Blue competent cells in accordance with the attached instruction manual (manufactured by Invitrogen). An ENOl R400-420 primer (5 '-GCAAGGGGCACC AGTCTTGATCTG-3 '(SEQ ID NO:48))was used to determine the nucleotide sequence. Every mutation clone plasmids with correct sequences were transformed to E.coli BL21 Rosseta. The ENOl mutation protein was expressed in E.coli by IPTG induction and purified by Ni-agarose in accordance with the attached instruction manual provided by the manufacturer (Qiagen) after the bacteria pellet was sonicated. The purity of each mutant protein was analyzed by 12% SDS PAGE.
[00161] To determine the binding activity of each mutant protein, 400ng/100microL of human ENOl protein or mutated ENOl protein was coated on a 96- well ELISA plate overnight at 4°Cand theplate was washed with PBS. The plate was blocked with 1% BSA (w/v) in PBS at room temperature for lhour,then washed again with lx PBS. A primary antibody (EN 10 mAb) was 2- fold serial diluted to 15 different concentrations and added to the plate at 37°C for lhour. After the reaction was done, the plate was washed 3 times with l x PBS. Al/8000 dilution of goat anti-mouse-HRP antibody was added and incubated at 37 °C for 1 hour, then the plate washed 3 times with l PBS. Then, TMB substrate was added and thereaction was allowed to proceed at room temperature for 30 minutes. The reaction was stoppedby adding IN HC1 and OD 450 was read to determine the activity. Each study was repeated three times. Data are presented as mean±SD.OD readings and concentrations of antibody were used to make a multiple scatterplotusingSigmaplot™. The Kd values were predicted by four parameter logistic fit.
[00162] According to the ENOl large portion deletion study resultsshown in Example 9, a peptidesequence
FDQDDWGAWQKFTASAGIQVVGDDLTVTNPKRIAKAVNEKS(SEQ ID NO:49) from the residuenumber 296 to336is required for tight binding of ENOl protein with EN10 mAb. "Tight binding" as used herein refers to binding between a specific binding agent (e.g., an antibody, an scFv or Fab fragment) and a ligand/target (e.g., a peptide, protein, or cell) with a dissociation constant(Kd) of 10 nM or lower, preferably 1.0 nM or lower.
[00163] The above deletion experiments identify residues 296 to 336 on ENOl as the region for the antibody binding. To further characterize the actual binding sites (e.g., epitopes),the crystal structure of ENOl was downloaded from protein data bank (pdb-entry: 2PSN) to analyze residue positions from this region. There are eleven amino acid residues including D300, W301, G302, Q305, K306, A309, K326, K330, N333, E334, and K335 exposed on the protein surface (FIG.13 A, putative epitope). Bysite-direct mutagenesis, these 11 amino acids were mutated and resulting mutant proteins were expressed mE.coli and purified, respectively(FIG.13B). Every purified ENOl mutant protein wasanalyzed for any Kdchanges(as compared withENOl binding)usingELISA.
[00164] Theresults indicate that there are three functional classes of amino acid residues in these mutants. Amino acid residuesW301 and K330 are importantfor the binding between ENOl protein and EN10 mAb. If these two amino acid residue are mutated to alanine, respectively, the binding activities of these two ENOlmutants to EN10 mAb are significantly compromised. The second class of amino acid residues includesA309, E334, 335 andD300. If E334, K335 and D300are mutated to alanineorthe A309 is mutated to glycine,respectively, the binding activities of these ENOlmutants to EN 10 mAb are compromised. The rest of amino acid residues including G302, Q305, K306, N333, and K326 belong to the group of amino acids residues thathave no significant binding effects on ENOl protein binding toENIO mAb(FIG.13C and Table 2). Theseresults suggest that W301, K330A309, E334, K335 and D300 are important for the protein-protein binding between ENOl and EN10 mAb. These amino acid residues belong tosequences of ENOl peptide 1,296FD Q D D W G A W Q K F TA309(FIG.13D,SEQ ID NO:9) and peptide 2,326 K R I A K A V N EK S336 (FIG.13D,SEQ ID NO:10 ), which may be thebinding epitopes of EN10 mAbin the amino residue number from 296 to 335( FIG.13D; SEQ ID NO:49) of human ENOl .
Table2: Mutant Kd Values
Mutant KD Mutant KD Mutant KD
Wild type 4.43±0.65 x 10"10 A300 6.33±2.57 x 10"8 A301 Non-detected
A302 3.45±0.28 x 10"10 A305 3.31±0.50 x 10"10 A306 4.65±2.42 x 10"10
A309 5.63±2.67 x 10~9 A326 7.87±1.35 x 10"10 A330 Non-detected
A333 4.13±1.09 x 10"10 A334 1.07±4.39 x 10"7 A335 9.57±3.72 x 10"9
Example 14
Structure analysis of ENOl and EN 10 mAb
[00165] The crystal structure of Enolase alpha was downloaded from protein data bank (pdb-entry: 2PSN). The Fab fragment structure of EN10 mAb was built by thehomology modeling. The first step wasto find a template structure with high sequence similarity to the EN10 mAb.The NCBI database (www.ncbi.nlm.nih.gov.) was searched and many highly homologous hits were found.The heavy-chain framework (pdb-entry: 3DGG) (identity 89%) and the light chain framework (pdb-entry: 1F6L) (identity 95%) were chosen. The antibody homology model of EN 10 mAb was constructed with the programs Accelrys Discovery Studio. ENOl and antibody structures were used for docking. In order to alleviate the computational load, only the CDR loops of variable domain of the antibodies were docked to ENOl. The framework of antibody was fixed. Since the docking procedure explores a relative large area around the starting position, very careful initial positioning of the docking partners was not required. When using ZDock, the structures were perturbed with x-y-z axis movements (perturbation along the line of centers, in angstroms perturbation in the plane perpendicular to the line of centers, in angstroms-rotational perturbation, in degrees) and approximately 5000 poses were generated for each docking run. Clustering analysis of the ZDock results was conducted with both a 5 A and ΙθΑ cut-off. The interaction residues of ENOl were chosen for epitope mapping.
[00166] 3D structure of human ENOl protein is shown in FIG.14A. Amino acidresidues W301, K330,300D, E334, AND K335 are labeled. The critical residues in plasminogen binding site of ENOl protein published by Wang et al. are indicated(250-FFRSGKY 256). FIG.14B shows that the binding complex of ENOl and EN10 mAb is predicted about 5 A to 15 A close to the plasminogen binding site of ENOl. The gap between plasminogen binding site and EN10 mAb is far less than 34 A, which is the molecular sized of human plasminogen. Thisstudy suggests that the binding of ENOl and EN 10 mAb shields the plasminogen binding site of ENOl, prevents the access of plasminogen to ENOl, results in compromising the plasminogen receptor activity of ENOl, and prevents cell dissociation from extracellular matrix.
Example 15
Biacore Analysis
[00167] Biacore analysis using a soluble ENOl protein to bind antibodies immobilized on CM5 chips was performed for EN10 mAb to estimate their affinity for soluble antigen.
[00168] The mouse variable region and human Fc chimera antibody expression vector pTCAE8-EN01,as shown in FIG.15 A, was introduced into host cells to prepare recombinant antibody-expressing cells. As the host cells for expression, the FreeStyle293 cells (manufactured by Invitrogen) were used. The vector was introduced into the host cells bylipofectamine 2000in accordance with the attached instruction manual (manufactured by Invitrogen.) About 2.5 microgram of the antibody expression vector was linearized by restriction enzymes, the gene was introduced into 4 l06 cells, and cells were inoculated to a6-well culture plate. The agent corresponding to a selection marker of the expression vector was added, and cells were continuously cultured to form a stable pool. [00169] A culturesupernatant containing human IgG antibody was prepared by the method described below. The antibody-producing cells was acclimated in aFree style™293 Expression Medium (GIBCO). Thecellswere cultured in a tissue culture flask, and the culture supernatant was collected when the viable rate of the cells was 90%. The collected supernatant was filtered through 10 micrometer and 0.2 micrometer filters (manufactured by Millpore) to remove contaminants. The culture supernatant containing the antibody was affinity-purified using Protein A (manufactured by Millipore), PBS as an absorption buffer, and 20 mM sodium citrate buffer (pH 3.0) as an elution buffer. The elution fractions were adjusted to around pH 6.0 by adding 50 mM sodium phosphate buffer (pH 7.0). The prepared antibody solution was replaced with PBS using a dialysis membrane (10,000 MW cut, manufactured by Spectrum Laboratories) and filter-sterilized through a membrane filter (manufactured by Millpore) having a pore size of 0.22 micrometer to yield the purified antibody. The concentration of the purified antibody was determined by measuring the absorbance at 280 nm and converting the measured value based on 1.45 optimal density equaling 1 mg/ml.
[00170] For binding kinetics, surface plasmon resonance (SPR) measurement with a BIAcore 2000 (BIAcore, Inc., Piscataway, N.J.) was used as previously described (Karlsson&Falt, (1997) J. Immunol Methods 200:121-133). Carboxymethylated dextran biosensor chips (CM5, BIAcore Inc.) were activated with N-ethyl-N'-(3 -dimethylaminopropyl)-carbodiimide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) according to the supplier's instructions. ChimeraEN 10 mAb was diluted with 10 mM sodium acetate, pH 4.8, into 5microgram/ml before injection at a flow rate of 20 micro L/minute to achieve approximately 100 response units (RU) of coupled protein followed by the injection of 1M ethanolamine to block unreacted groups. For kinetics measurements, two-fold serial dilutions of ENOl (0.3125nM to 40nM) were injected in HBS-PBiacore running buffer provided by the manufacturer (BIAcore, Inc., Piscataway, N.J.) at 25degree C at a flow rate of 25 microL/min, and binding responses on the EN10 mAb were corrected by subtraction of responses on a blank flow cell.Association rates (kon or ka) and dissociation rates (koff or kd) were calculated using a simple one-to-one Langmuir binding model with separate fittings of kon and koff was used. (BIAcore.TM. Evaluation Software version 3.2).
Table 3
Figure imgf000039_0001
Figure imgf000040_0001
[00171] The resultsareshown in the FIG.15B and Table 3. Thekon and koff of EN10 mAbbinding withENOl are 3.57xl05 and 8.271x10"5,respectively, and Kdis 2. 311 + 0.003x10"10mol/L. The Kdanalyzed by SPR is very close to that of binding ELISA in the Example 2.
Example 16
Inhibitory effect of the EN 10 antibody on tumor growth in the presence of complement.
[00172] The EN10 mAb has a goodaffinity with Kdabout 2.31 I±0.003xl0"10mol/L and a potential for the further development. To evaluate the therapeutic effects of EN 10 mAb, a CLl-5 mouse xenograft model was performed. CL1-5F4 lung adenocarcinoma cells (l xl06cells/mouse; 5mice/group) were subcutaneously inoculated at Day 0. The therapeutic procedure was performed 14 days after the tumor inoculation by administrating lOmpk (mg/Kg) of an isotype control (CTL), EN 10 mAb, or Erbitux™ antibody twice per week. The rabbit baby serum was given 2h post-injection. The tumor volume and bodyweight of each mouse was measured weekly. Data arerepresented as mean ± SD for individual groups.
[00173] The results are shown in FIG.16. After 14 days the tumor sizesof mice bearing CLl-5 are about 30 to 100 mm3, and there are no significant tumor size differences among the control group, EN 10 mAb, and Erbitux treatment groups after day 25. After day 29, the tumor of mice in the control group starts to grow exponentially, and there are no significant tumor growths in the Eribitux™ and EN 10 mAb treatment mice. After day 36, the average tumor size of the control group mice is 4330±990mm3 (N=5), and for mice treated with lOmpk ofENIO mAb and Erbitux, the average tumor sizes are 358.6±240mm3 (N=5) and 219 + 98mm3 (N=5),respectively. The average tumor sizes of both EN 10 mAb and Erbitux™treatment groups aresignificantly smaller, as compared to that of the control group with a P value of 0.004 and 0.003, respectively. There are no significant tumor size difference tween EN10 mAb and Erbitux™ treatment groups (P=0.41).This result indicates that EN10 mAb and Erbitux have the tumor growth inhibitionactivity on CLl-5 cells when combined with complement C3 in the mouse xenograft model and EN 10 mAb has a good efficacy as a reagent for cancer therapy.
Example 17
Blockade of tumor spread of pancreatic caners in spleen-liver metastatic colony formation assay.
[00174] Results fromExamples 2 and 3 suggest that administration of ENOl antibody can attenuate the invasion and metastasis of cancer cells and results from Example 16 indicates that ENOl mAb has efficacy ininhibiting the tumor growth when combined with complement. To evaluate the anti-metastatic activity of EN 10 mAb on cancer cells in vivo, a spleen-liver metastatic assay were performed. PDAC1 pancreatic adenocarcinoma cells (l xl06/mouse; 5 mice/group) were injected into spleen. Then, 10 mpk of EN10 mAb and IgG control mice were administrated into each group by IP, respectively. The mice were scarified 6 weeks after tumor inoculation. The number of tumor nodules in livers and the weight of organs, including lungs, spleens, livers, pancreas, and kidneys, of each mouse were counted and measured, respectively. Data are represented as mean ± SD for individual groups.
[00175] Results are shownin FIGs. 17 A, 17B, and 17C. When liver tumor nodules and volumes are compared, the average tumor nodules and volumes of control IgG treated group are 34±10 (N=3) and 8500+3500 (N=5), respectively.However, as the same background mice were treated with 10 mpk of EN 10 mAb, the average tumor nodules and volumes of treated mice are 10±4 (N=3) and 500+120 (N=5), respectively. There are significant tumor nodule number and volume differencesbetween 10 mpk ENOl and the control IgG treated groups (P<0.05)( FIG 17A and 17B). When average weights of organs were compared between the 10 mpk ENOl and the control IgG treatment groups, there are no significant organ weight differences, including lungs, spleens, pancreases, and kidneys. However,mice treated with lOmpk IgG has anaverage liver weight about 7.2+3g (N=5), which is statistic different, ascompared to that of mice treated with lOmpkENIO mAb (1.6±0.2g(N=5), P<0.05). Theseresults suggest that administration of EN 10 mAb inhibits the metastasis of PDAC1 cells from primary tumor site to the liver.
[00176] In sum, EN10 mAb uses its ENOl plasminogen receptor antagonistactivity to inhibitthe plasminogen activation, thereby inducing down regulation of protease activity on the cell surface, which in turn results in the inhibition of dissociation of cancer cells from extracellular matrix. As a result, antibodies against ENOl caninhibitthe invasion capability of cancer cells. Thesedata support that ENOl antibodies (e.g., EN10 mAb) have favorable affinity, efficacy and potential as a therapeutic antibodyfor thetreatment of cancers.
[00177] In addition to inhibiting cancer dissociation from extracellular matrix and, therefore, inhibiting cancer invasion, an antibody of the invention may also be used to diagnose a cancerous condition. As noted above and shown in FIG. 1 A and FIG. IB, cancer cells express ENOl on cell surfaces, while normal cells do not. Therefore, one can detect antibody binding to cell surface ENOl as an indication of cancerous conditions.
[00178] For example, in accordance with embodiments of the invention, a test sample may be reacted with an anti-ENOl antibody (e.g., EN10 mAb) and the binding of the antibody to the sample may be determined, such as with ELISA, fluorescence cell sorting, fluorescence microscope, or any suitable method known in the art. If the binding is detected or is above a threshold value (e.g., a reference value for a normal sample), then one may conclude that the sample contains cancer cells. [00179] While the invention has been described with respect to a limited number of embodiments, those skilled in the art, having benefit of this disclosure, will appreciate that other embodiments can be devised which do not depart from the scope of the invention as disclosed herein. Accordingly, the scope of the invention should be limited only by the attached claims.

Claims

CLAIMS What is claimed is:
1. An antibody, or an scFv or Fab fragment thereof, comprising: one or more sequences selected from the group consisting of HCDR1 (GYTFTSCVMN; SEQ ID NO:3), HCDR2 (YINPYNDGTKYNEKFKG; SEQ ID NO:4), HCDR3 (EGFYYGNFDN; SEQ ID NO:5), LCDR1 (RASENIYSYLT; SEQ ID NO:6), LCDR2 (NAKTLPE; SEQ ID NO:7), and LCDR3 (QHHYGTPYT; SEQ ID NO:8).
2. The antibody, or the scFv or Fab fragment thereof, according to claim 1, comprising a heavy chain that comprises the sequences of HCDR1 (GYTFTSCVMN; SEQ ID NO:3), HCDR2 (YINPYNDGTKYNEKFKG; SEQ ID NO:4), and HCDR3 (EGFYYGNFDN; SEQ ID NO:5).
3. The antibody, or the scFv or Fab fragment thereof, according to claim 2, wherein the heavy chain comprises the sequence of SEQ ID NO:l .
4. The antibody, or the scFv or Fab fragment thereof, according to claim 1, comprising a light chain that comprises the sequences of LCDR1 (RASENIYSYLT; SEQ ID NO:6), LCDR2 (NAKTLPE; SEQ ID NO:7), and LCDR3 (QHHYGTPYT; SEQ ID NO:8).
5. The antibody, or the scFv or Fab fragment thereof, according to claim 4, wherein the light chain comprises the sequence of SEQ ID NO:2.
6. The antibody, or the scFv or Fab fragment thereof, according to claim 1 , comprising a heavy chain that comprises the sequences of HCDR1 (GYTFTSCVMN; SEQ ID NO:3), HCDR2 (YINPYNDGTKYNEKFKG; SEQ ID NO:4), and HCDR3 (EGFYYGNFDN; SEQ ID NO:5) and a light chain that comprises the sequences of LCDR1 (RASENIYSYLT; SEQ ID NO:6), LCDR2 (NAKTLPE; SEQ ID NO:7), and LCDR3 (QHHYGTPYT; SEQ ID NO:8).
7. The antibody, or the scFv or Fab fragment thereof, according to claim 6, wherein the heavy chain comprises the sequence of SEQ ID NO:l and the light chain comprises the sequence of SEQ ID NO:2.
8. An antibody, or an scFv or Fab fragment thereof, wherein the antibody, or the scFv or Fab fragment thereof, can bind to a peptide or protein comprising the sequence of FDQDDWGA WQKFTA (SEQ ID NO:9) or KRIAKAVNEKS (SEQ ID NO: 10) with a dissociation constant (¾) of 10 nM or lower.
9. The antibody, or the scFv or Fab fragment thereof, according to claim 8, wherein the peptide or protein comprises the sequence of
FDQDDWGAWQKFTASAGIQVVGDDLTVTNPKRIAKAVNEKS (SEQ ID NO:49).
10. The antibody, or the scFv or Fab fragment thereof, according to any one of claims 1-9, wherein the antibody is a monoclonal antibody.
11. A pharmaceutical composition for treating lung, breast, pancreas, liver, colorectal, or prostate cancer, comprising the antibody or the scFv or Fab fragment thereof, according to any one of claims 1-10.
12. A method for diagnosis of cancer, comprising:
reacting a sample from a test subject with the antibody, or the scFv or Fab fragment thereof, according to any one of claims 1-10 to determine an extent of binding; wherein the extent of binding is greater than a reference value indicates a cancerous condition.
13. A method for inhibiting cancer invasion, comprising:
administering to a subject in need thereof the antibody, or the scFv or Fab fragment thereof, according to any one of claims 1-10.
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AU2013408259A1 (en) 2016-08-04
AU2013408259B2 (en) 2020-05-21
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RU2016129166A (en) 2018-01-25

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