TW201125580A - Anti- α -enolase I antibodies for diagnosis and treatment of α -enolase I-associated diseases - Google Patents

Abstract

The invention relates to antibodies against α -enolase I, their pharmaceutical compositions and diagnosis and treatment uses. Particularly, the invention provides polyclonal anti- α -enolase I antibodies and monoclonal single-chain variable fragment (scFv) anti- α -enolase antibodies, pharmaceutical compositions containing the same and their uses in uses in diagnosis and treatment of cancers, autoimmune disorders, ischemia and bacterial infection.

Description

201125580 VI. Description of the Invention: [Technical Field] The present invention relates to an anti-α-isoenolase 1 (or rice cultivar, α-isoenolase) antibody, a pharmaceutical composition thereof, and a diagnosis and treatment thereof use. In particular, the present invention provides multiple strains of anti-"isokinase! Antibody and single-stranded single-stranded fragment (scFv) anti-α-isololase antibody, pharmaceutical composition containing the same, and use for direct diagnosis and treatment of cancer, autoimmune disease, ischemia and bacterial infection . [Prior Art] Cancer is characterized by an abnormality in the normal tissue or an increase in the number of neoplastic cells, which form a tumor mass, into the adjacent tissues, and the production of malignant cells, which eventually (4) blood or: The bar system spreads to the local lymph nodes and spreads to distant sites via a process called metastasis. The identification of these tumor-associated cell surface antigens has produced the ability of specific filaments to cancer cells to be destroyed via antibody-based therapies. In an attempt to discover cancer therapies or effective cell counts, researchers have managed to discern peptides that are specifically overexpressed on the surface of specific types of cancer cells compared to - or multiple normal non-cancer cells. In other attempts to discover effective cellular targets for cancer therapy, researchers have sought to identify polypeptides produced and secreted by certain types of cancer cells that are expressed in a higher amount than that produced by or - a variety of normal non-cancer cells. The above-mentioned confirmed progress has been made in mammalian cancer therapy, but it is highly desirable to separately detect the presence of tumors in mammals and other diagnostic agents and therapeutic agents for treating cancer. ' 143761.doc 201125580 Isoenolase was originally described as an enzyme involved in glycolytic metabolism and catalyzing the conversion of 2-phosphoglycerate to phosphoenolpyruvate. In mammals, isoenolase has three isoforms, called α-ΕΝΟΙ, β-ΕΝ03, and γ-ΕΝ02. Α-isoenolase is the main form of isoenolase, which is present in the early stages of embryonic development and is commonly found in various types of tissues, while γ-ΕΝ02 and β-ΕΝ03 are found only in neurons and muscle cells ( Antikainen et al, 2007, FEMS Immunol Med Microbiol 51, 526-534; Chang et al, 2006, Clin Cancer Res 12, 5746-5754). Information on the nucleic acid and amino acid sequences of a-isoenolase is available on the NCBI website at http://www.ncbi.nlm.nih.gov/UniGene/clust.cgi? ORG=Hs&CID = 517145. Isoenolase is reported to be a multifunctional protein exhibiting enzyme catalysis, structure and receptor function (Chang et al., 2006, Clin Cancer Res 12, 5746-5754; Lee et al., 2003, Arthritis Rheum 48, 2025-2035 ). In addition to its glycolytic function, alpha-isoenolase has been found to play an important role in a variety of biological and pathophysiological processes. In particular, α-isoenolase is thought to play an important role in tumor formation. This protein is found on the cell surface and acts as a plasminogen receptor that can play a role in tumor invasion (Redlitz et al., 1995, Eur J Biochem 227, 407-415). Alpha-isoenolase has been previously regulated in a variety of highly tumorigenic or metastatic cell lines (Chang et al, 2006, Clin Cancer Res 12, 5746-5754; Peebles et al, 2003, Carcinogenesis 24, 65 1 -657; Satoshi Ito, 2007, Cancer Science 98, 499-505; Wu et al, 2002, Clin Exp Metastasis 19, 319-326; Zhang et al, 2000, J Surg Res 93, 143761.doc 201125580 108-119) . Overexpression of α-isoenolase is associated with tumorigenicity leading to multiple types of cancer, indicating its pathophysiological role in cancer formation (Altenberg and Greulich, 2004, Genomics 84, 1014-1020). Furthermore, autoantigens of X-isoenolase have been identified in non-small cell lung cancer, and their overexpression is highly correlated with poor survival outcomes (Chang et al, 2006, Clin Cancer Res 12, 5746-5754). In addition to its role in cancer, alpha-isoenolase has been implicated in a number of diseases, including autoimmune disorders, ischemia and bacterial infections. (Antikainen et al., 2007, FEMS Immunol Med Microbiol 51, 526-534; Bogdanos Et al. 2004, J Autoimmune Dis 1, 4; Gitlits et al, 2001, J Investig Med 49, 138-145; Jiang et al, 1997, Cancer Res 57, 5328-5335; Kinloch et al, 2005, Arthritis Res Ther 7, R1421-1429; Saulot et al., 2002, Arthritis Rheum 46, 1196-1201) 〇 Therefore, a-isoenolase is a therapeutic agent for the treatment or prevention of cancer development or a potential marker for detecting cancer There is still a need to detect, treat, prevent, and reverse the development of cancer. SUMMARY OF THE INVENTION One object of the present invention is to provide a multi-strain of avian anti-α-isoenolase I that specifically binds α-isoenolase I. Another object of the present invention is Provided is a purified monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain immunoglobulin variable domain and a light chain immunoglobulin variable domain which binds to α-isoenolase I, wherein the light chain immunoglobulin is Γ The variable domain comprises the following amino acid sequence: (i) SGGSGSYG (SEQ ID 143761.doc 201125580 ID NO: 1), SGGSSSYGYG (SEQ ID NO: 2), SGSSSGSYG (SEQ ID NO: 3), SGGSSSYGYS (SEQ ID) in CDR1 NO: 4) or SGSSGYGYG (SEQ ID NO: 5); (ii) ANTNRPS (SEQ ID NO: 6), NDNQRPS (SEQ ID NO: 7), RDDKRPS (SEQ ID NO: 8), SNNQRPS (SEQ) in CDR2 ID NO: 9) or SNDDRPS (SEQ ID NO: 10); and (iii) GGYDSSAGI (SEQ ID NO: 11), GSGDSSTGM (SEQ ID NO: 12), GSGESSTNNGI (SEQ ID NO: 13), GSMDSSNSGV in CDR3 (SEQ ID NO: 14) or GGYDSSASYVGI (SEQ ID NO: 15); and wherein the heavy chain immunoglobulin variable domain comprises the following amino acid sequence: (i) SFNMF (SEQ ID NO: 16) in CDR1, SHDMG (SEQ ID NO: 17), DYCVQ (SEQ ID NO: 18), SFYMF (SEQ ID NO: 19) or SYAMH (SEQ ID NO: 20); (ii) GINNAGSTTNHGAAVKG (SEQ ID NO: 21) in CDR2 ' GIENAAGIGTFYGAAVKG (SEQ ID NO: 22) ' AISN TGRYTGYGSAVKG (SEQ ID NO: 23) > GISGDGRYTGYGAAVDG (SEQ ID NO: 24) or GISRDGGSSTRYYGAAVKG (SEQ ID NO: 25); and (iii) SPGGIDGIDG (SEQ ID NO: 26), GADTGGWPAANIDA (SEQ ID NO: 27) 'DGCAGCCGSYYIDG (SEQ ID NO: 28) 'ESGSGCCNGDNIDA (SEQ ID NO: 29) or DSDNGGYYCDDIDA (SEQ ID NO: 30). Another object of the present invention is to provide a pharmaceutical composition comprising the polyclonal antibody of the present invention and a pharmaceutically acceptable carrier. Another object of the present invention is to provide a method for treating or preventing an α-isoenolase I-related disorder comprising administering a therapeutically effective amount of the 143761.doc 201125580 strain antibody or monoclonal antibody of the present invention. Another object of the present invention is to provide an in vitro diagnostic method for detecting the presence of isobeolase I in a sample, which comprises: (1) contacting a sample with the anti-α-isoenolase I antibody of the present invention; (ii) detecting the formation of a complex between the α-isoenolase anti-aa and the sample. Another object of the present invention is to provide a kit for detecting the presence of α-isoenolase I in a sample comprising the anti-cardinolase I antibody of the present invention or a fragment thereof, and optionally Information materials. [Embodiment] The present invention generates and characterizes multiple strains of anti-isoenol-antibody and single-stranded variable-chain fragment of a single chicken by a phage display system (hearts such as isoamylase I antibody. These antibodies may have Helps to develop molecular diagnostics and therapeutics for cancer, autoimmune disorders, ischemia and fine infections. Definitions It should be noted that the term anti-isoenolase antibody bodies. Thus, the term "is used interchangeably. And "at least one" as used herein, as used herein, The "Notes", "Eight, _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ A substance that is generally ambiguous to interfere with the diagnostic or therapeutic use of the polypeptide, and may include enzymes, hormones, and other eggshell or non-protein solutes. As used herein, the term "枋a" refers to the ability to combine Targeted intact antibody or antibody fragment. sentence put p ( c including Fv, ScFv, Fab• and F(ab,)2, single 143761.doc 201125580 strain and multiple antibodies, engineered antibodies (including chimeric antibodies, CDR grafts and humanized antibodies, fully human antibodies, and labor) Selection of antibodies), and synthetic or semi-synthetic antibodies produced using steroid rendering or alternative techniques. Small fragments such as Fv and scFv have advantageous properties for diagnostic and therapeutic applications due to their small size and consequent excellent tissue distribution. The term "antibody" is further intended to encompass antibodies, digested fragments, specific portions and variants thereof, including antibody mimetics or antibody portions comprising mimicking the structure and/or function of a particular fragment or portion thereof, including single chain antibodies and Fragments. Functional fragments include antigen-binding fragments that bind to alpha-isoenolase. For example, the invention encompasses antibody fragments capable of binding to cardinolase or a portion thereof, including but not limited to Fabs (eg, Produced by papain digestion), Fab' (for example, by pepsin digestion and partial reduction) and F(ab,) 2 (for example, by pepsin elimination) Generated), (e.g., by plasmin digestion generated), pFcI (e.g., by pepsin or plasmin digestion generated), Fd (e.g., by pepsin

Different formulations, conventional (multi-strain) antibodies of different antibodies have high specificity for a single antigenic site. This is a single determinant on different antibodies to different determinants (antigenic determinants). Each monoclonal antibody is directed against the antigen on a single 143761.doc 201125580. Thus, the modifier "single plant" indicates the characteristics of an antibody obtained from a population of antibodies from a substantially homologous population and should not be construed as requiring any particular method. Produce antibodies. For example, monoclonal antibodies for use in accordance with the present invention can be prepared by the fusion tumor method known in the art, or can be prepared by recombinant methods such as those described in U.S. Patent No. 4,816,567. For example, "monoclonal antibodies" can also be isolated, for example, from phage libraries generated using the techniques described in McCafferty et al, Nature, 348:552-554 (199). The term "specifically binds" generally means that an antibody binds to an epitope via its antigen-binding domain and binds to an antigen-binding domain and an epitope; there is some complementarity. According to this definition, when an antibody binds to an epitope via its antigen-binding domain, it is easier to bind a random unrelated epitope than the antibody. The antibody is said to "specifically bind" to the epitope. The term "specificity, as used herein, to define the relative affinity of an antibody for binding to an epitope" as used herein, as used herein, refers to both therapeutic and prophylactic therapies. Lights § My "cancer" and "cancerous or characterized are usually cells ^ ° 35 physiological conditions. Examples of cancer include (but not) cancer, lymphoma, blastoma, sarcoma and leukemia. More specific examples of such itch include squamous itch, small cell lung cancer, non-small cell glandoma, gastrointestinal cancer, sputum D. cancer, glioma cell, A, M, Field tumor, cervical cancer, egg field

Cancer, hepatocellular carcinoma, itching "4, monthly cancer, bladder cancer, cancer, colorectal cancer, intrauterine / 143761.doc 201125580 cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, Vulvar cancer, thyroid cancer, liver cancer and various types of head and neck cancer. The term "individual" or "animal" or "patient" or "mammal" means any individual, particularly a mammalian subject, in need of diagnosis or treatment. Mammalian animals include humans, livestock, farm animals, and zoos, athletics, or pet animals such as dogs, I seedlings, guinea pigs, rabbits, rats, mice, horses, cows, cows, and the like. Antibody The present invention relates to an antibody which specifically binds to an α-isoolase polypeptide, an antigen-binding antibody fragment thereof, and an antibody variant and a segment. Such antibodies may be, for example, multiple strains or monoclonal antibodies. More preferably, it is a monoclonal antibody. Still more preferred are chimeric or humanized antibodies, and still better as human antibodies. Multi-Strain Antibodies In one aspect, the present invention provides a multi-strain antibody against avian anti-α-isoenolase that specifically binds to cardinolase. In an embodiment of the invention, . The hai poultry is chicken and the multi-strain antibody is a plurality of anti-α-isoenolase Υ Υ antibody. The multi-antibody antibody is preferably an anti-α-isoenolase 隹匕丫 antibody. A plurality of antibodies are known in the art. First, multiple injections of the human heart isoenolase I polypeptide are used to immunize avian species (eg, chickens, ducks, turkeys, and their jaws) to cause an immunogenic response in the bird. Time to collect serum or eggs from avian mice immunized with α_isoenolase I after forming a high-valence anti-α-isoenolase 1 antibody in poultry. The immunological avian serum and yolk contain many natural exposures. The antigen is specific and the antibody is used. The next step of the 5H method is to isolate the IgY-dissolved wound of 14376l.doc 201125580 phage globulin from serum or egg yolk (the lysate is known to contain α-isochelinase specificity). Antibody) This can be achieved by using the egg yolk of a commercial product such as Promega Corporation's EGGstract® IgY Purification System (Promega Corporation, Madison, Wis. USA) and HiTrapTM IgY Purified HP Column (GE Healthcare, USA). Isolation and immunity from egg yolk Other methods of globulin, such as other sequential precipitation methods, are well known to those skilled in the art (see, for example, Sc〇pes, R κ "Protein Purification: Principles and Practice", Springer_

Verlag New York, 1994, its teachings on protein purification methods are incorporated herein by reference). Fully satisfactorily implementing the present invention, "the protein is dissolved, this system

The conventional protein separation method is "salting out" 4 by precipitating the protein from the salt solution, and isolating the IgY antibody from the avian gold. In addition, the method.

Monoclonal antibody I4376I.doc 201125580 In another aspect, the present invention provides a purified monoclonal antibody or antigen-binding fragment thereof comprising 〇t-isololase I, comprising a heavy chain immunoglobulin variable domain and light A chain immunoglobulin variable domain, wherein the light chain immunoglobulin variable domain comprises the following amino acid sequence: (i) SGGSGSYG (SEQ ID NO: 1), SGGSSSYGYG (SEQ ID NO: 2), SGSSGSYG in CDR1 (SEQ ID NO: 3), SGGSSSYGYS (SEQ ID NO: 4) or SGSSGYGYG (SEQ ID NO: 5); (ii) ANTNRPS (SEQ ID NO: 6), NDNQRPS (SEQ ID NO: 7) in CDR2, RDDKRPS (SEQ ID NO: 8), SNNQRPS (SEQ ID NO: 9) or SNDDRPS (SEQ ID NO: 10); and (iii) GGYDSSAGI (SEQ ID NO: 11), GSGDSSTGM (SEQ ID NO: 12) in CDR3 , GSGESSTNNGI (SEQ ID NO: 13), GSMDSSNSGV (SEQ ID NO: 14) or GGYDSSASYVGI (SEQ ID NO: 15); and wherein the heavy chain immunoglobulin variable domain comprises the following amino acid sequence: (i) SFNMF (SEQ ID NO: 16), SHDMG (SEQ ID NO: 17), DYCVQ (SEQ ID NO: 18), SFYMF (SEQ ID NO: 19) or SYAMH (SEQ ID NO: 20) in CDR1; GINNAGSTTNHGAAVKG in CDR2 (SEQ ID NO: 2) 1) > GIENAAGIGTFYGAAVKG (SEQ ID NO: 22) ' AISNTGRYTGYGSAVKG (SEQ ID NO: 23), GISGDGRYTGYGAAVDG (SEQ ID NO: 24) or

GISRDGGSSTRYYGAAVKG (SEQ ID NO: 25); and (iii) SPGGIDGIDG (SEQ ID NO: 26) in CDR3 ' GADTGGWPAANIDA (SEQ ID NO: 27), DGCAGCCGSYYIDG (SEQ ID NO: 28), ESGSGCCNGDNIDA (SEQ ID NO: 29 ) or DSDNGGYYCDDIDA 143761.doc 201125580 (SEQ ID NO: 30).

In one embodiment, the purified monoclonal antibody or antigen-binding fragment thereof of the present invention comprises a framework region selected from the VH* and VL FR1, FR2, FR3 and FR4 framework regions encoded by the germline gene sequence of chicken immunoglobulin (about VL and * VH, please refer to www.ncbi.nlm.nih.gov/nuccore/16902088 and http://www.ncbi.nlm.nih.gov/sites/entrez?cmd=Retrieve&db=protein& dopt= GenPept&RID=MS2P2YSH012&log%24=prottop&blast_rank=l &list_uids=104726) or the amino acid sequence and at least the VH and VL FR1, FR2 and FR3 framework regions encoded by the germline gene sequence of chicken immunoglobulin 85% of the same framework region. Preferably, the purified antibody or antigen-binding fragment thereof comprises an amino acid sequence and at least 87%, 88%, 89%, 90% of the VH and VL FR1, FR2 and FR3 framework regions encoded by the germline gene sequence of chicken immunoglobulin, 91%, 92%, 93%, 94%, 95%, 97%, 98% or 99% identical framework regions. In certain embodiments, the VH and VL domains of the monoclonal antibodies of the invention can be germlined, or the framework regions (FR) of such domains can be altered using conventional molecular biology techniques. Multiple locations (eg, at least 70%, 80%, 85%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94./〇 or 95%, 97%, 98%) Or 99% of the 'framework position' matches the common amino acid sequence of the human germline gene or human germline gene product. In other embodiments, the framework sequence is different from the reproductive system. In one embodiment, the purified monoclonal antibody or antigen-binding fragment thereof of the present invention comprises a VL complementarity determining region having an amino acid sequence 选自 selected from the group consisting of: i) SEQ ID NO in CDR1 : l ' CDR2 ^ 13 143761.doc 201125580 SEQ ID NO: 6 and SEQ ID NO: 11 in CDR3; ii) SEQ ID NO: 2 in CDR1, SEQ ID NO: 7 and CDR3 in CDR2 SEQ ID NO: 12; iii) SEQ ID NO: 3 in CDR1, SEQ ID NO: 8 in CDR2 and SEQ ID NO: 1 3 in CDR3; iv) SEQ ID NO: 4, CDR2 in CDR1 SEQ ID NO: 14 in SEQ ID NO: 9 and CDR3, SEQ ID NO: 5 in CDR1, SEQ ID NO: 10 in CDR2 and SEQ ID NO: 15 in CDR3, and having The VH complementarity determining region of the amino acid sequence of the following amino acid sequence: i) SEQ ID NO: 16 in CDR1, SEQ ID NO: 21 in CDR2 and SEQ ID NO: 26 in CDR3; ii) SEQ ID NO: 17 in CDR1, SEQ ID NO: 22 in CDR2 and SEQ ID NO: 27 in CDR3; iii) SEQ ID NO: 18 in CDR1, SEQ ID NO: 23 and CDR3 in CDR2 SEQ ID NO: 28; iv) CDR1 SEQ ID NO: 19, SEQ ID NO: 24 in CDR2 and SEQ ID NO: 29 and v in CDR3 SEQ ID NO: 20 in CDR1, SEQ ID NO: 25 in CDR2 and SEQ ID NO in CDR3 : 30. Preferably, the VL complementarity determining region has the amino acid sequence mentioned in ii) or v) and the VH complementarity determining region has the amino acid sequence mentioned in ii) or v). In another embodiment, the purified monoclonal antibody or antigen-binding fragment thereof of the present invention comprises a light chain immunoglobulin variable domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 1-35 and having The heavy chain immunoglobulin variable domain of the amino acid sequence of the group consisting of SEQ ID NO: 36-40 is selected. In one embodiment, the CDR sequences of the antibodies or fragments thereof are only insubstantially different from the CDR sequences of the antibodies described herein. Non-substantial differences include less amino acid changes than 143761.doc -14-201125580, such as one or two amino acid substitutions in any of the 5-7 amino acids in the CDR (eg, Chothia or Kabat CDR) sequence. . Typically, the amino acid is substituted with an associated amino acid having similar charge, hydrophobicity or stereochemical properties. Such substitutions are within the skill of those of ordinary skill. Unlike in CDRs, more substantial changes can be made to the structural framework regions (FR) without adversely affecting the binding properties of the antibodies. Alterations to FR include, but are not limited to, humanizing or engineering non-human frameworks for certain framework residues that are important for antigen contact or stabilization of binding sites, for example, changing the type or subclass of constant regions, changing Specific amino acid residues that may alter the effector function (such as Fc receptor binding) (Lund et al, (1991) J. Immunol. 147: 2657-62; Morgan et al, (1995) Immunology 86: 319-24), or change the species derived from the constant region. The monoclonal antibodies or antigen-binding fragments thereof of the present invention can be obtained by phage display technology. The bacterium cell presentation involves localization of the peptide in a form fused to the end of the sheath protein (e.g., pill, pIIV) of phage particles. See Scott, J. K. and G. P. Smith (1990) Science 249 (4967): 386-390; and Lowman, Η. Β· et al., (1991) Biochem. 30 (45): 10832-10838. In general, a polypeptide having a specific binding function is isolated by incubation with a target, washing off the non-binding phage, dissolving the bound sputum, and then cultivating the sputum population by infecting the fresh bacterial culture. Amplification. Other presentation formats and methods include mRNA presentation, ribosome or polysome display, eukaryotic virus presentation, and bacterial, yeast, and mammalian cell surface presentation. See Mattheakis, LC et al., (1994) Γ PNAS USA 91 (19): 9022-9026; Wilson, DS et al., '143761.doc -15· 201125580 (2001) PNAS USA 98(7): 3750-3755; Shusta, EV et al, (1999) Curr. Opin. Biotech. 10(2): 117-122; and Boder, ET and KD Wittrup (1997) Nature Biotech. 15(6): 553-557. A variety of alternative presentation techniques for presentation on the surface of microorganisms have been developed and reported as general strategies for isolating protein binding peptides, but have not been reported successfully. See Maurer, J. et al., (1997) J. Bacteriol. 179(3): 794-804; Samuelson, P. et al., (1995) J. Bacteriol. 177(6): 1470-1476; Robert, A Et al., (1996) FEBS Letters 390(3): 327-333; Stathopoulos, C. #Λ, (1996)Αρρ1·Microbiol. & Biotech· 45(1-2): 112-119 ; Georgiou, G Et al., (1996) Protein Engineering 9(2): 239-247; Haddad, D. et al. (1995)? Ugly: \18 11111111111〇1.&]^(11.31^^(;1 *〇1^〇1.12(3- 4): 175-186; Pallesen, L. et al., (1995) Microbiol. 141(Pt 11): 2839-2848; Xu, Z· and SY Lee (1999) Appl. Environ. Microbiol. 65(11): 5142-5147; Wernerus, H. and S_ Stahl (2002) FEMS Microbiol, Lett. 212(1): 47-54;

Westerlund-Wikstrom, B. (2000) Int. J. Med. Microbiol. 290(3): 223-230 ° The multi-plant or monoclonal antibodies described herein may also be labeled with detectable or functional markers. Detectable labels include radioactive labels such as ηι or 99Tc, which can be linked to the antibodies described herein using chemical methods known in the art. The label also includes an enzyme label 'such as horseradish peroxidase or alkaline phosphatase. The label further includes a chemical moiety, such as biotin, which can be detected by binding to a homologous detectable moiety (e.g., labeled avidin) 143761.doc -16·201125580. The binding properties of the antibodies disclosed herein can be measured by any suitable method, including the following methods: Biacore analysis, enzyme-bound immunosorbent assay (ELIS A), X-ray crystallography, sequence analysis, and scanning mutation induction, and are well known in the art. Other methods. The pharmaceutical composition of the present invention may be incorporated into a pharmaceutical composition, such as in combination with a pharmaceutically acceptable carrier, and may also contain, for example, various diluents, fillers, salts. Buffers, stabilizers, solubilizers, and other materials well known in the art. The term "pharmaceutically acceptable" means a non-toxic substance that does not interfere with the biologically active efficacy of the active ingredient. The nature of the carrier can be determined by the route of administration. The term "therapeutically effective amount" as used herein means that the various active ingredients of a pharmaceutical composition or method are sufficient to demonstrate meaningful benefit to the patient (eg, to ameliorate the symptoms of the condition, to treat the condition, or to ameliorate the condition The total amount of 疥 旬 。. When applied to individual active ingredients administered separately, the term refers to a single component. When applied to a combination, the term refers to the combined amount of active ingredients that produce a therapeutic effect, regardless of In combination, sequential or simultaneous administration. In the practice of the exemplary therapeutic methods or uses described herein, a therapeutically effective amount of a combination of alpha-isoenolase can be administered (4) tomb... 'functional ^isoenolase 1 The antibody that the letter = complex forms (and, for example, neutralizes or inhibits one or each of the relevant activities), is administered to an individual, such as a mammal (e.g., human sputum can be used alone or in combination with other therapies according to the methods described. 舍, 143,761. Doc 201125580 - or when multiple agents are administered together, antibodies can be administered simultaneously with the second agent, or separately (eg, sequentially). If administered separately (eg, sequentially), the attending physician will decide to vote Appropriate sequence of body and other agents. Administration of pharmaceutical compositions (e.g., pharmaceutical compositions containing antibodies that bind to alpha-isoenolase) can be carried out in a variety of conventional manners, such as oral ingestion, inhalation, or via Intradermal, subcutaneous or intravenous injection. Subcutaneous administration of the patient is preferred. When a therapeutically effective amount of the combination of α-isoenol, such as and interferes with the formation of functional α•isoenolase I signaling complex, is orally administered. In the meantime, the binding agent will be in the form of a sachet, a powder, a solution or a medicinal agent. When administered as a keying agent, the pharmaceutical composition may additionally contain a solid carrier such as gelatin or a: a tablet, a capsule, and The powder contains from about 5% to 95% binder, and preferably from about 25% to 9G% binder. When administered in liquid form, a liquid carrier such as an oil of water, petroleum, animal or vegetable origin may be added (such as Peanut oil, mineral oil, large oil or sesame oil) or synthetic oil. The liquid form of the pharmaceutical composition may further comprise a physiological saline solution, dextrose or other saccharide solution, or such as ethylene glycol, propylene glycol or polyethylene glycol. Glycols When the therapeutically effective amount of the combination of the isoenolase I is administered by intravenous, transdermal or subcutaneous injection, the binding agent will be in the form of a pyrogen-free, non-enteral extractable water solution. Such parenterally acceptable protein solutions (having predetermined pH, isotonicity, stability, and the like) are known in the art. They are used for intravenous, percutaneous or subcutaneous injection. Preferably, the pharmaceutical composition should contain an isotonic vehicle in addition to the binding agent, such as sodium chloride smear, Linyi injection (r (four) ringing. ^, right 143761.doc -18- 201125580 Main injection, dextrose and sodium chloride injection, lactated Ringer's injection back J or other vehicles known in the art. Pharmaceutical compositions may also contain stabilizers, preservatives, buffers, antioxidants Or other additives known to the skilled artisan. The amount of antibody in a pharmaceutical composition will depend on the nature and severity of the condition being treated and the nature of the prior treatment that the patient has experienced. Ultimately, the attending physician will determine the amount of antibody used to treat each individual patient. Initially, the attending physician will administer a low dose of antibody and observe the patient's response. A larger dose of antibody can be administered until the optimal therapeutic effect is obtained for the patient, and at this time no further dose increase is generally made. For example, the dose can be administered in the range of 0.1-50 mg per kg body weight, 0.5-50 mg ' 1-100 mg, 〇 5_25 mg, 〇 15 mg or 1-8 mg. Therapeutic and prophylactic use of an anti-α-isoenolase 1 antibody The antibody of the present invention can be used for the treatment of an α-isoenolase I-related disorder, for example, a condition selected from one or more of the following: cancer, autoimmune disorder, Ischemia and bacterial infection. Alpha-isokinase I and its receptors may be involved in the development of at least some types of cancers, such as 'cancers derived from ash-producing cells or cancers derived from lung cancer. The cancer is preferably lung cancer, breast cancer, anal cancer, bladder cancer, bone cancer, intestinal cancer, brain tumor, kidney cancer, leukemia, liver cancer, pancreatic cancer, prostate cancer, and rectal cancer. Lung cancer is more preferably non-small cell lung cancer. Cancer refers to the loss of reactivity to normal growth control, or a variety of cells, and usually proliferate as regulation decreases (relative to the corresponding normal cells). Diagnostic use of anti-α-isoenolase I antibody [143761.doc 19 201125580] In another sad example, the present invention provides an in vitro method for detecting the presence of α-isoenolase I (eg, biological samples) (such as organization) biopsy). The method comprises: (1) contacting the sample with the anti-α-isoenol antibody of the present invention, and (η) detecting the formation of a complex between the enrichment and the sample by the α-isoenolase. This may include contacting a reference sample (eg, a control sample) with a ligand, and determining the extent of complex formation between the ligand and the sample relative to the & reference sample. Composites in the sample or individual relative to the control sample or individual Changes in the formation of the substance (eg, statistically significant changes) may indicate the presence of (X-isoenolase I in the sample. It can be detected by measuring or visualizing the isoenol-bound ligand or unbound ligand). Complex formation between "isoenolase!antibody and alpha-heterologous enzyme. A conventional detection assay such as enzyme-binding immunosorbent assay (ELISA), radioimmunoassay (RIA) or tissue immunization can be used. Histochemistry. In addition to labeling α-isoenol _; E antibody, it is also possible to analyze the α-isotype in the sample by competitive immunoassay using the standard of the label detectable substance and the unlabeled α-isoenol oxime antibody. The presence of dilute alcoholase I. Kit anti-isoxanolase! antibody or The segment can be provided, for example, as a kit component: in a kit. For example, a 'set includes (a galenolase! antibody or a guanidine fragment, for example, including an anti-alpha-isoenol antibody or a fragment thereof) And (b) informational material as appropriate. The informational material may be described by the method and/or anti-alpha_isoenol, antibody or fragment thereof for: descriptive, instructional, sales or other The form of the poor information material of the kit is not limited. In the embodiment, the information material I4376I.doc •20· 201125580 may include information on the production of the compound, the molecular weight of the compound, the concentration, the effective period, the batch, or Production location information, etc. In one embodiment, the informational material relates to the use of a ligand to treat, prevent, or diagnose a condition as described herein. The kit may include one or more for the combination of the ancient and the old for 3 - a container of an isoenolase I antibody or a combination of two. In some embodiments, the kit contains individual compartments 11, spacers or compartments for use in the group of poor families. Can be included in bottles, small bottles or syringes, and information materials ^ In a plastic envelope or package, in other embodiments, the kits are each packaged in an early-un-separated container. For example, the composition is contained in a bottle with a poorly-formed material in the form of a logo, small In a syringe or a syringe, in some of the dimples, the kit includes a plurality (eg, a pack) of individual containers, each container: one or more unit dosage forms of an anti-alpha iso-livalase 1 antibody or fragment thereof (eg, The dosage form described herein. For example, the kit includes a plurality of injection crying, ampoules, drop bags, nebulizers or inhalation devices each containing a unit dose of an anti-heart dismutase 1 antibody or fragment thereof, Or multiple unit doses. The t group includes, as appropriate, devices suitable for administration of the composition, such as syringes, sputum, recordings, spoons, droppers (eg, eye drops), swabs (eg, cotton swabs or Wood Pharmaceuticals sign), or any such transfer device. In a preferred embodiment, the device is an implantable device that dispenses a dose of ligand. The following examples are provided to aid the understanding of the invention, but are not intended to be construed as limiting the scope of the invention in any way. Example 1 characterization of recombinant α-isoenolase and multiple anti-α-isoglycolase [s 143761.doc -21 · 201125580

IgY antibody by reverse transcription-PCR, using gene-specific primer 5·-GGTGGAATTCTATCTATTCTCAAGATCCATGCC-3\^q ID NO·· 41) (pre-priming) and 5,-ACTCCATGGTTAC7TCGCC4y4GGGG77TCT-3' (SEQ ID NO: 42) Inverse primer) A gene encoding an α-isoenolase protein was selected from PE089 cells. The resulting PCR fragment was cloned into the EcoRI and Ncol sites in the pGEX-KG vector and transformed into E. coli BL-21 (DE3) strain for its expression. This gene was also subcloned into the pET21a vector using 5'-CCGCGTGAATTCGGGGATCCATGTCTATTCTCAAGATCC-3, (SEQ ID NO: 43) (pre-priming) and 5'-CATGGAGTCGACCTCGXGCTTGGCCAAGGGGTTTCTG-3' (SEQ ID NO: 44) (reverse primer). And expressed in the form of His fusion α-isoenolase. Individual pure lines were grown overnight in 5 ml of LB medium containing ampicillin (100 pg/ml) at 37 °C. The bacterial culture was diluted 10-fold with the same LB medium and allowed to grow further until OD6G() reached between 0.6 and 1_0. To induce GST fusion α-isoenolase or His fusion α-isoenolase protein expression, add isopropyl-β-D-thiogalactopyranoside (IPTG) until its final concentration in culture It is 0.5 mM. The cell pellet was resuspended in 2 ml of 1x Triton X-100 in lxPBS and thawed in three rounds (-70 ° C) and thawed (37. lysed. After centrifugation, the resulting cell lysate was according to the manufacturer. Description (General Electronics, Piscataway, NJ, USA) was incubated with glutathione Sepharose 4B or Ni2+-charged resin column γ to purify the α-isoenolase protein. 143761.doc -22 - 201125580 After electrophoresis and Coomassie blue staining, the purified His fusion α-isoenolase and GST fusion α-isoenolase appeared as a 48kD and 75kD monochromatic band (Fig. 1A Lanes 2 and 3). As shown in lane 3 of Figure 1B, the GST-fused α-isoenolase was confirmed using an anti-GST antibody. Similarly, it was produced in a chicken immunized with purified His fusion α-isoenolase. Multiple IgY antibodies can clearly recognize the His-fused α-isoenolase immobilized on Western blots and GST-fused α-isoenolase (lanes 2 and 3 in Figure 1C). Example 2 Chicken Immunity by Muscle Intra-injection, purified at 100 pg in an equal volume of Freund's complete adjuvant Alpha-isosalination enzymes make white leghorns (house chickens (Ga//&) immunization. Three additional immunizations with incomplete adjuvants at 7-day intervals. Thereafter, IgY antibodies in serum and yolk were collected and titrated by enzyme-linked immunosorbent assay (ELISA) to determine the presence of a humoral anti-α-isoenolase immunoreactivity as previously described (Akita and Nakai, 1993a, b) The egg yolk was separated from the egg white using 10% dextran sulfate for IgY purification. The purified total IgY antibody from each egg was dissolved in 5 ml of TBS containing 0.05% sodium azide and stored at -20 °C. The serum and print of chicken were collected before and after each immunization. The total IgY antibody in the yellow was purified and the anti-chicken IgY antibody combined with horseradish peroxidase was used to detect the presence of heavy and light chain fragments (data not shown). The binding activity of α-isoenolase immobilized on the pores of nitrocellulose membrane (Fig. 1C) or ELISA plate was tested using purified IgY. As shown in Fig. 2, prepared from egg yolk after the fourth immunization. IgY antibody specifically binds α-isosaturated at a titration of 1:16,000 [143761.d Oc -23- 201125580 Alcoholase, not bovine serum albumin, indicates a strong humoral antibody response in chicken hosts. In contrast, IgY antibodies from pre-immune chicken eggs exhibit minimal binding signals to both antigens. Example 3 Construction and Biopanning of the scFv Antibody Library Establishment of an antibody library based on previous reports: Andris-Widhopf, J.,

Rader, C., Steinberger, P., Fuller, R., Barbas, C.F., 3rd edition > 2000, Methods for the generation of chicken monoclonal antibody fragments by phage display. J Immunol Methods 242, 15 9-181. Briefly, chicken spleens were collected after final immunization and immediately homogenized for 7 days in Trizol reagent. 10 pg of total RNA was reverse transcribed into the first strand using the Superscript RT kit (Invitrogen, USA). After amplification with chicken-specific factors, the PCR products of the heavy and light chain variable (VH and VL) regions are subjected to a second round of PCR using short or long linkers to form a full-length scFv fragment, which is further digested and colonized. In the pComb3X vector. Recombinant DNA was transformed into E. coli XL-1 blue strain by electroporation. Recombinant bacillus was produced by the addition of VCS-M13 to assist the bacillus to 4 〇/. Polyethylene glycol 8000 and 3% NaCl (w/v) were precipitated and finally resuspended in phosphate buffered saline (pbS) containing 1% bovine serum albumin (BSA) and stored at 4 °C. Then, 1011 plaque_forming units (pfu) of recombinant sputum cells from the scFv antibody library were added to the wells pre-coated with α-isoenolase protein (0.5 μg/well), and at 37 ^ Cultivate for 2 hours. After removal of unbound phage, the bound phage was eluted with 〇丨ΜHC1/glycine (pH 2.2)/0.1% BSA, neutralized with 2 M Tris buffer and used to infect XL-1 blue strain. The amplified bacillus was collected as described above, especially 143761.doc •24·201125580, and recovered for the second round of selection. Repeat the panning process three or four times. A set of pure lines were randomly selected and grown using the final panning process. After 6 hours of incubation at 0.5 mM IPTG, bacterial cells were harvested and resolved by three rounds of freezing; lysate was treated with East and/or sonication. The scFv antibody expression of the supernatant and its binding reactivity to cardinolase were analyzed using Western blotting and ELIS A. ScFv antibodies were also prepared in flow cytometry and immunofluorescence assays. These antibodies were expressed in TOP 10F' E. coli (Invitr〇gen' non-inhibitory strain) and used as described by the manufacturer (Amersham Biosciences, UK). Ni2+-charged agarose purification ° Chickens were sacrificed 8 weeks after the last immunization and total RNA was extracted from the swollen spleens to construct an antibody library. The full length scFv gene fragment was amplified using 2 consecutive PCR steps. In the first PCR, the VH gene product was amplified to a size of 400 bp using a primer containing a short linker (GGSSRSS) (SEQ ID NO: 45) and a long linker (GGSSRSSSSGGGGSGGGG) (SEQ ID NO: 46), respectively As shown by EnVH.S (Fig. 3A, lane 2) and EnVH.L (Fig. 3A, lane 3). Accordingly, the VL gene was amplified into a 350 bp band and loaded in lane 4 (EnVL) in Figure 3A. Subsequently, the amplified VH was ligated with VL to form a full-length scFv gene fragment of about 750 bp as shown by EnscFv.S (lane 2) and EnscFv.L (lane 3) in Figure 3B. A plurality of sputum strains were constructed to present antibody libraries and used to screen for specific anti-α-isoenolase scFv antibodies. Example 4 Characterization of anti-α-isoenolase scFv pure line To detect the expression of scFv antibody, the cell lysate was subjected to sodium dodecyl sulfate sodium polyacrylamide gel electrophoresis (SDS-PAGE). All proteins were transferred to 143761.doc -25-201125580 onto a stone xiaohua cellulose membrane (Amersham Biosciences, UK) and subsequently blocked with 5% skim milk TBST solution for 1 hour. A goat anti-chicken IgY light-chain antibody (Bethyl Laboratories, Montgomery, TX, USA) diluted 1:3000 was added and incubated for an additional hour. The membrane was washed three times with TBST for 5 minutes each time. Bound antibodies were detected by the addition of 1:3000 dilution of horseradish peroxidase (HRP) in combination with donkey anti-goat Ig antibody (Sigma, St. Louis, MO, USA). After three washes, the film was developed with diaminobenzidine (DAB) until the desired strength was achieved. To study their binding reactivity, purified IgY from a fourth immunized chicken or a scFv-expressing antibody was incubated with purified alpha-isoenolase immobilized on a nitrocellulose membrane or ELIS A plate well, and subsequently borrowed It was detected by adding a goat anti-chicken IgY light chain antibody as described above and HRP-conjugated donkey anti-goat Ig antibody. Each sample was subjected to an ELISA test in double replicate wells. Four rounds of panning were performed as described above. After each panning, 15 pure lines were randomly selected and the 750 bp fragment insert and its scFv antibody protein expression in the pCom3_X selection vector were analyzed. Information (not shown) indicates that the pure lines of each round of panning are 67% (10/15), 87% (13/15), 100% (15/15) and 100% (15/15) with full length inserts. In addition, 10 pure lines with 750 bp inserts from the last round of panning were induced to express their scFv antibodies. As shown in Figure 4, goat anti-chicken IgY light chain antibody and horseradish peroxidase (HRP) were combined with donkey anti-goat Ig antibody to detect pure line EnL2, EnL4, EnL5, EnL6 and EnL7 to |J scFv Performance, with little or no performance detected for pure EnLl, EnL3, EnL8, EnL9 and EnL 1 0. These results indicate that the amount of immunoglobulin genes with highly conserved sequences can vary significantly, even under the same experimental conditions of 143761.doc -26-201125580. Example 5 Gene sequencing and ELISA analysis using ompseq (5'-AAGACAGCTATCGCGATTGCAGTG-3') (SEQ ID NO: 47) and HRML-F by an automated sequencer (ABI PRISM 377; Perkin-Elmer, National Health Research Institute) (5'-GGTGGTTCCTCTAGATCTTCC-3') (SEQ ID NO: 48) The primers were subjected to nucleotide sequence determination of the heavy and light chain variable regions of the selected pure line. The results were analyzed using the alignment program BLAST and the vector NTI (http://www.ncbi.nlm.nih.gov/BLAST). The nucleotide sequences of the variable regions of the 10 pure heavy and light chain genes were determined and aligned with the germline gene sequences of chicken immunoglobulin. The results revealed that 30% of the sequenced lines (EnL5, EnL6 and EnL7) share the same heavy and light chain genes, resulting in similar profiles of their scFv antibodies, as seen in lanes 5, 6 and 7 of Figure 4. The overall mutation rate compared to the germline gene sequence was 18.6°/ in the heavy and light chain variable regions, respectively. To 27.4% and 13.5% to 23.1% (Figure 5). The binding activity of the £11 B1 to £111^7 3〇?丫 antibody against 〇1-isoenolase was analyzed using £1^13. These scFv antibody fragments were found to exhibit significant binding activity against alpha-isoenolase compared to two other scFv antibodies previously characterized and known to specifically recognize the SARS-CoV spike protein. In particular, EnL2, EnL4, EnL5 and EnL6 scFv antibodies exhibited stronger positive reactivity than purified multiple IgY from chickens immunized with human alpha-isoenolase (Figure 6(A) and B)). Example 6 Flowcytometry Analysis PE089 cell line was originally obtained from a 36 year old patient with stage IV lung adenocarcinoma. 143761.doc -27- 201125580 fluid tumor cells from the National Institute of Dr. Neng-Yao Shih from Cancer Research, National Health Research Institutes, Tainan, Taiwan. These cells have been cultured in vitro for at least 40 passages in RPMI 1640 supplemented with 5% fetal calf serum, 2 mmol/L branic acid and antibiotics. A total of 2χ106 cells were collected and fixed with 2% paraformaldehyde as previously described. The α-isoenolase expressed in PE089 cells was detected with purified scFv EnL2 and EnL5 antibodies, and mouse anti-HA (1:200) and Cy-2 goat anti-mouse antibody (1:200) ( Jackson ImmunoResearch Laboratories, West Grove, PA, USA) was visualized and analyzed using a FAC Scan flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). Negative control analysis was performed as described above without using the scFv EnL2 and EnL5 antibodies once, and a positive control analysis was performed using rabbit anti-human isoenolase antibody (1:200) instead of scFv EnL2 and EnL5 antibodies. To test the binding reactivity of these selected scFv antibodies, the human alpha-isoenolase gene was transfected into ΡΕ089 tumor cells for expression on the membrane surface, followed by flow cytometry. Purified EnL2 and EnL5 scFv antibodies (data not shown) on SDS-PAGE are capable of detecting recombinant α-isoenolase protein expressed in PE089 cells, binding signals and α-isoenolase The binding signal of the specific rabbit polyclonal antibody of specificity was similar, as shown in FIG. Example 7 Immunofluorescence and Laser Scanning Microscopy ΡΕ089 cells (2 x 10 5 cells/ml) were seeded on coverslips and incubated on ice by using an equal volume of ice-cold 8% paraformaldehyde (freshly prepared). Minutes 143761.doc -28- 201125580 Fixed. After fixation, the cells were dehydrated by sequential treatment with 70%, 95% and 99% sterol and rehydrated with 95% and 70% sterols. The slides were then covered with blocking buffer (lx PBS containing 1% BSA) for 1 hour at room temperature. After washing with lxPBS, the scFv antibody was incubated with the cells for an additional hour at room temperature. Finally, the binding of the scFv antibody to the α-isoenolase protein was detected by mouse anti-HA antibody and Cy-2 goat anti-mouse antibody in combination. The nuclei were also comparatively stained using the recommended sputum solution (Invitrogen, USA). The slides were observed using a Confocal Spectral Microscope Imaging System (TCS SP5, Leica). Immunocytochemical staining was also used to assess the binding ability of the purified EnL2 and EnL5 scFv antibodies to the alpha-isoenolase molecules expressed in PE089 cells. The alpha-isoenolase molecule has been shown to be predominantly expressed and located on the nuclear membrane of ΡΕ089 cells (communication with Dr. N-Y Shih). Thus, recombinant EnL2 and EnL5 scFv antibodies exhibited significant binding signals around the nuclear membrane, as shown in Figure 8. In contrast, two negative controls, including goat anti-mouse antibodies that bind to the Cy-2 or 4L8 pure line (scFv antibodies that are specific for SARS-CoV spiny protein), did not exhibit any reactivity. Cell morphology and distribution under light microscopy are included in the leftmost panel for comparison. Taken together, the results further indicate that phage display technology can be a better alternative to the selection and production of anti-specific antigen scFv antibodies. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a graph showing the expression of recombinant α-isoenolase and a plurality of anti-α-isoenolase IgY antibodies. The samples in each figure are protein label (lane M), purified GST (lane 1), purified α-isoenolase (lane 2), and purified GST-α-isoenolase fusion egg [143761.doc -29- 201125580 White (Ice 3) The purified protein (Fig. a) developed by Coomassie blue staining was spotted on nitrocellulose paper, and anti-cafe antibody (Fig. B) or the fourth immunized chicken serum (Fig. B) Figure C) Detection. The molecular weight of the recombinant α-isoenolase protein was about 48 kD; Figure 2 shows the humoral response in chickens after the fourth immunization by ELIS A analysis. The purified α-isoenolase protein and bovine serum antigen (bsa) were applied to the wells. The specific binding activity of a series of diluted IgY antibodies to a-isoenolase or BSA was examined. The solid and open bars indicate the binding of IgY to cardioenolase in the 4th immunized chicken or pre-immune chicken, respectively. In addition, the binding of IgY to BSA in the 4th immunized chicken or pre-immune chicken indicated by dark gray and light gray bars was displayed as a negative control in parallel; Figure 3 is the pcR of the variable region in the chicken immunoglobulin gene. Amplification. The light chain (EnVL) and the heavy chain with a short linker (EnVHS) or the heavy chain with a long linker (EnVH.L) were successfully amplified (panel A). The second round of pcR produces a full-length scFv* factor fragment (EnseFvS) with a ligated linker or a full-length scFv gene fragment with a long linker (EnscFv L) (Fig. b); Figure 4 shows the analysis by Western blotting method 3 ( Bubo antibody performance. The same amount of total cell lysate from % pure lines was loaded onto SDS_pAGE and transferred to nitrocellulose paper. The goat anti-chicken light chain antibody and HRP binding were diluted with 丨:3〇〇〇 in turn. The anti-goat IgG detects the presence of the scfv antibody. The predicted molecular weight of the scF^ segment is approximately 35 kDa. The ink dot is a representative result of the scFv expression in the selected pure line; Figure 5 is the scFv antibody pair analyzed by ELISA. Purification of the binding activity of α-isoenolase. Purification of the 4th round of panning randomly selected cells containing scFv antibody 143761.doc •30- 201125580 Lysate and purification on plate wells...isoenolase Binding. The goat anti-chicken light chain antibody diluted with 1··3000 was used in sequence, and HRp-conjugated anti-goat was used to detect the binding activity and measured at 450 rnn. Two anti-SARS_CoV scFv antibodies were used (SC〇S-S8). And SC〇S-L22) as a negative control. Do not add a reconstitution s In the case of the cFv antibody, another control experiment was performed as described. A plurality of IgY antibodies of chickens immunized with purified α-isoenolase were used as positive controls. The ELISA data were expressed as the average of the double replicate experiments; Sequence analysis of VL (Fig. 6 (A)) and VH (Fig. 6 (B)) sequences of scFv antibodies. Ten pure VH and VLi nucleotide sequences were determined and translated into amino acid sequences to interact with chicken germline genes. Amino acid sequence alignment. FR: framework region; CDR: complementarity determining region. Sequence gaps are introduced to maximize alignment and are represented by blank regions. Dots indicate common sequences. Framework regions (FR) and complementarity determining regions ( The CDR) borders are indicated above the germline sequence; Figure 7 is the binding activity of the scfv antibody to the purified isoenolase as analyzed by 'Guap cell cytometry. Purified EnL2 & EnL5 scFv antibody, mouse anti-HA ( 1:200) and Cy-2 combined with goat anti-mouse antibody (1:2〇〇) to detect α•isoenolase bound to PE089 cells. Gray thin line indicates negative control, which was treated with DMS0 alone and passed through firefly. Light-labeled cells stained with surface-labeled Abs; gray solid lines indicate the cursor The cells stained with the isotype control were recorded; and the solid black line indicates cells stained with anti-surface α_isoenolase scFv antibody EnL2 and EnL5 fluorescently labeled Abs. One of three independent experiments was shown. The results; and Figure 8 is the immunofluorescence staining of the α-isoenolase protein in PE089 cells. The cells were fixed to glass plates as described in Materials and Methods [b 143761.doc -31. 201125580. The a-isoenolase expression was detected using purified EnL2 and EnL5 scFv antibodies followed by mouse anti-HA and Cy-2 binding goat anti-mouse antibodies. The nucleus (red) appears by PI. Both EnL2 and EnL5 scFv antibodies clearly stained the PE089 cell nuclear membrane (green). The anti-SARS-CoV scFv antibody 4L8 did not exhibit any reactivity with the nuclear envelope. 143761.doc •32· 201125580 Sequence Listing <110>»Zombie University<120>Anti-α-isoenolase antibody for diagnosis and treatment of α-isoolase-related diseases <130〉 T54267/US1358 <160> 50 <170> Patentln version 3. 5 <210> 1 <211> 8 <212> PRT < 213 > artificial sequence < 220 < 223 > 223 > synthetic sequence <400> 1

Ser Gly Gly Ser Gly Ser Tyr Gly <210〉 2 <211> 10 <212> PRT <213>Artificial Sequence <220><223> EnL2 VL CDR1 <400> 2

Ser Gly Gly Ser Ser Serrr Gly Tyr Gly 15 10 <210> 3 <211> 8 <212> PRT < 213 > 213 > Artificial Sequence <220 < 223 > 223 > VL of EnL3 CDK1 <400> 3

Ser Gly Ser Ser Gly Ser Tyr Gly 1 5 <210> 4 <211> 10 <212> PRT <213>Artificial Sequence <220> <223> EnL4 VL CDR1 <400> 4

Ser Gly Gly Ser Ser Ser Tyr Gly Tyr Ser 1 5 10 <210> 5 <211> 9 <212> PRT <213>Artificial Sequence <220> 143761.doc 201125580 <223> EnL5 VL CDRl <400〉 5

Ser Gly Ser Ser Gly Tyr Gly Tyr Gly 1 5 <210〉 6 <211> 7 <212> PRT <213>Artificial Sequence <220><223> EnLl VL CDR2 <400> 6

LAC2 <220〉 7

Asn Asp Asn Gin Arg Pro Ser <210> 8 <211> 7 <212> PRT <213>Artificial Sequence <220> <223> EnL3 VL CDR2 <400>

Arg Asp Asp Lys Arg Pro Ser 1 5 <210> 9 <211> 7 <212> PRT <213>Artificial Sequence <220><223> EnL4 VL CDR2 <400>

Ser Asn Asn Gin Arg Pro Ser 1 5 <210> 10 <211> 7 <212> PRT <213>Artificial Sequence <220> <223> EnL5 VL CDR2 <400>

Ser Asn Asp Lys Arg Pro Ser 143761.doc 201125580 1 5 <210> 11 <211> 9 <212> PRT <213>Artificial Sequence <220> <223> EnLl VL CDR3 <400&gt ; 11

Gly Gly Tyr Asp Ser Ser Ala Gly He 1 5 <210> 12 <211> 9 <212> PRT <213>Artificial Sequence <220> <223> EnL2 VL CDR3 <400> 12

Gly Ser Gly Asp Ser Ser Thr Gly Met 1 5 <210> 13 <211> 11 <212> PRT <213> Artificial sequence <220><223> EnL3 VL CDK3 <400> 13

Gly Ser Gly Glu Ser Ser Thr Asn Asn Gly lie 1 5 10 <210> 14 <211> 10 <212> PRT <213> Artificial Sequence <220><223> EnL4 VL CDR3 <400> 14 1 5 <210> 15 <211> 12 <212> PRT <213> Artificial sequence <220><223> VL3 of VL of EnL5 <400>

Gly Ser Met Asp Ser Ser Asn Ser Gly Val

Gly Gly Tyr Asp Ser Ser Ala Ser Tyr Val Gly He 1 5 10 <210> 16 <211> 5 143761.doc 201125580 <212> PRT <213> Artificial Sequence <220> <223> EnLl CDR1 <400> 16 Ser Phe Asn Met Phe 1 5 <210> 17 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> LV1 <223>;400> 17 Ser His Asp Met Gly 1 5 <210> 18 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> LR1 <400> of VH of EnL3 Asp Tyr Cys Val Gin 1 5 <210> 19 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> EnL4 VH CDR1 <400> 19 Ser Phe Tyr Met Phe 1 5 <210> 20 <211> 5 <212> PRT <213> Artificial sequence <220><223> EnL5 VH CDR1 <400> 20 Ser Tyr Ala Met His 1 5 &lt ;210> 21 <211> 17 <212> PRT <213> Artificial sequence <220><223> EnLl VH CDR2 143761.doc 201125 580 <400〉 21

Gly lie Asn Asn Ala Gly Ser Thr Thr Asn His Gly Ala Ala Val Lys 15 10 15

Gly <210> 22 <211> 18 <212> PRT < 213 > 213 > artificial sequence <220 < 223 > 223 & CDR 2 of VH of EnL2 <400 > 22

Gly lie Glu Asn Ala Ala Gly lie Gly Thr Phe Tyr Gly Ala Ala Val 15 10 15

Lys Gly <210> 23 <211> 17 <212> PRT <213>Artificial sequence <220><223> LV2 <400> of VH of EnL3 23

Ala lie Ser Asn Thr Gly Arg Tyr Thr Gly Tyr Gly Ser Ala Val Lys 15 10 15

Gly <210> 24 <211> 17 <212> PRT <213> Artificial sequence <220><223> EnL4 VH CDR2 <400>

Gly lie Ser Gly Asp Gly Arg Tyr Thr Gly Tyr Gly Ala Ala Val Asp 15 10 15

Gly <210> 25 <211> 19 <212> PRT < 213 > 213 > artificial sequence <220 < 223 > LR 2 of VH of EnL5 <400> 25 143761.doc 201125580

Gly lie Ser Arg Asp Gly Gly Ser Ser Thr Arg Tyr Tyr Gly Ala Ala 15 10 15

Val Lys Gly <210> 26 <211> 10 <212> PRT <213>Artificial sequence <220><223> EnLl VH CDR3 <400> 26

Ser Pro Gly Gly lie Asp Gly lie Asp Gly 1 5 10 <210> 27 <211> 14 <212> PRT <213>Artificial Sequence <220> <223> EnL2 VH CDR3 <4 〇0> 27

Gly Ala Asp Thr Gly Gly Trp Pro Ala Ala Asn lie Asp Ala 1 5 10 <210> 28 <211> 14 <212> PRT <213>Artificial Sequence <220><223> EnL3 VH CDR3 <400> 28

Asp Gly Cys Ala Gly Cys Cys Gly Ser Tyr Tyr lie Asp Gly 15 10 <210> 29 <211> 14 <212> PRT <213>Artificial Sequence <220> <223> EnL4 VH CDR3 <400> 29

Glu Ser Gly Ser Gly Cys Cys Asn Gly Asp Asn lie Asp Ala 1 5 10 <210> 30 <211> 14 <212> PRT <213>Artificial Sequence <220> <223> EnL5 VH CDR3 <400〉 30

Asp Ser Asp Asn Gly Gly Tyr Tyr Cys Asp Asp lie Asp Ala -6- 143761.doc 201125580 5 10 <210> 31 <211> 102 <212> PRT <213>Artificial Sequence <220> < 223>EuLl Amino Acid <400> 31

Ala Leu Thr Gin Pro Ser Ser Val Ser Ala Asn Pro Gly Gly Thr Val 15 10 15

Glu lie Thr Cys Ser Gly Gly Ser Gly Ser Tyr Gly Trp Phe Gin Gin 20 25 30

Lys Ala Pro Gly Ser Ala Pro Val Thr Pro He Tyr Ala Asn Thr Asn 35 40 45

Arg Pro Ser Asp lie Pro Ser Arg Phe Ser Gly Ser Lys Ser Gly Ser 50 55 60

Thr Ala Thr Leu Thr lie Thr Gly Val Gin Ala Asp Asp Glu Ala Val 65 70 75 80

Tyr Phe Cys Gly Gly Tyr Asp Ser Ser Ala Gly lie Phe Gly Ala Gly 85 90 95

Thr Thr Leu Thr Val Leu 100 <210> 32 <211> 104 <212> PRT < 213 > 213 > 221 > 223 < 223 > 223 > LL > LL <400>

Ala Leu Thr Gin Pro Ser Ser Val Ser Ala Asn Pro Gly Gly Thr Val 15 10 15

Glu lie Thr Cys Ser Gly Gly Ser Ser Ser Tyr Gly Tyr Gly Trp Tyr 20 25 30

Gin Gin Lys Ser Pro Gly Ser Pro Pro Val Thr Val lie Tyr Asn Asp 35 40 45

Asn Gin Arg Pro Ser Asp lie Pro Ser Arg Phe Ser Gly Ala Leu Ser 50 55 60

Gly Ser Thr Ala Thr Leu Thr lie Thr Gly Val Gin Ala Asp Asp Glu 65 70 75 80

Ala Val Tyr Phe Cys Gly Ser Gly Asp Ser Ser Thr Gly Met Phe Gly 85 90 95 143761.doc 201125580

Ala Gly Thr Thr Leu Thr Val Leu 100 <210> 33 <211> 104 <212> PRT < 213 > 213 > Artificial Sequence <220 < 223 > 223 > EnL3 VL Amino Acid <400> 33

Ala Leu Thr Gin Pro Ser Ser Val Ser Ala Asn Pro Gly Gly Thr Val 15 10 15

Glu He Thr Cys Ser Gly Ser Ser Gly Ser Tyr Gly Trp Tyr Gin Gin 20 25 30

Lys Ser Pro Gly Ser Ala Pro Val Thr Val lie Tyr Arg Asp Asp Lys 35 40 45

Arg Pro Ser Asp lie Pro Ser Arg Phe Ser Gly Ser Leu Ser Gly Ser 50 55 60

Thr Ala Thr Leu Thr lie Thr Gly Val Gin Ala Glu Asp Glu Ala Val 65 70 75 80

Tyr Phe Tyr Gly Ser Gly Glu Ser Ser Thr Asn Asn Gly lie Phe Gly 85 90 95

Ala Gly Thr Thr Leu Thr Val Leu 100 <210> 34 <211> 105 <212> PRT <213>Artificial Sequence <220><223>EnL4 VL Amino Acid <400> 34

Ala Leu Thr Gin Pro Ser Ser Val Ser Ala Asn Pro Gly Gly Thr Val 15 10 15

Glu He Thr Cys Ser Gly Gly Ser Ser Ser Tyr Gly Tyr Ser Arg His 20 25 30

Gin Gin Lys Ser Pro Gly Ser Ala Pro Val Thr Ala He Tyr Ser Asn 35 40 45

Asn Gin Arg Pro Ser Asp lie Pro Ser Arg Phe Ser Gly Ser Thr Ser 50 55 60

Gly Ser Thr Ala Thr Leu Thr lie Thr Gly Val Gin Ala Asp Asp Glu 65 70 75 80

Ala Val Tyr Phe Cys Gly Ser Met Asp Ser Ser Asn Ser Gly Val Phe 85 90 95 143761.doc 201125580

Gly Ala Gly Thr Thr Leu Thr Val Leu 100 105 <210> 35 <211> 106 <212> PRT <213>Artificial Sequence <220><223>EnL5 VL Amino Acid <400>; 35

Ala Leu Thr Gin Pro Ser Ser Val Ser Ala Asn Pro Gly Gly Thr Val 15 10 15

Glu lie Thr Cys Ser Gly Ser Ser Gly Tyr Gly Tyr Gly Trp Tyr Gin 20 25 30

Gin Lys Ser Pro Gly Ser Ala Pro Val Thr Val lie Tyr Ser Asn Asp 35 40 45

Lys Arg Pro Ser Asp lie Pro Ser Arg Phe Ser Gly Ser Ala Ser Gly 50 55 60

Ser Thr Ala Thr Leu Thr lie Thr Gly Val Arg Ala Glu Asp Glu Ala 65 70 75 80

Val Tyr Phe Cys Gly Gly Tyr Asp Ser Ser Ala Ser Tyr Val Gly lie 85 90 95

Phe Gly Ala Gly Thr Thr Leu Thr Val Leu 100 105 <210> 36 <211> 144 <212> PRT < 213 > 213 > 221 > 223 > 223 > 223 > 400> 36

Ala Val Thr Leu Asp Glu Ser Gly Gly Gly Leu Gin Thr Pro Gly Gly 15 10 15

Thr Leu Ser Leu Val Cys Lys Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30

Asn Met Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45

Ala Gly lie Asn Asn Ala Gly Ser Thr Thr Asn His Gly Ala Ala Val 50 55 60

Lys Gly Arg Ala Thr lie Ser Arg Asp Asn Gly Gin Ser Thr Leu Arg 65 70 75 80

Leu Gin Leu Asn Asp Leu Arg Ala Glu Asp Thr Gly Thr Tyr Tyr Cys 85 90 95 -9- 143761.doc 201125580

Ala Arg Ser Pro Gly Gly He Asp Gly He Asp Gly Trp Gly His Gly 100 105 110

Thr Glu Val lie Val Ser Ser Thr Ser Gly Gin Ala Gly Gin His His 115 120 125

His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His Amino acid of VH <4 (8) > 37

Ala Val Thr Leu Asp Glu Ser Gly Gly Gly Leu Gin Thr Pro Gly Gly 15 10 15

Ala Leu Ser Leu Val Cys Lys Ala Ser Gly Phe Asp Phe Ser Ser His 20 25 30

Asp Met Gly Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Phe Val 35 40 45

Ala Gly lie Glu Asn Ala Ala Gly lie Gly Thr Phe Tyr Gly Ala Ala 50 55 60

Val Lys Gly Arg Ala Thr lie Ser Arg Asp Asn Gly Gin Ser Thr Val 65 70 75 80

Arg Leu Gin Leu Asn Asn Leu Arg Pro Glu Asp Thr Gly Thr Tyr Phe 85 90 95

Cys Ala Arg Gly Ala Asp Thr Gly Gly Trp Pro Ala Ala Asn lie Asp 100 105 110

Ala Trp Gly His Gly Thr Glu Val lie Val Ser Ser Thr Ser Gly Gin 115 120 125

Ala Gly Gin His His His His His His His His Gly Ala Tyr Pro Tyr Asp Val 130 135 140

Pro Asp Tyr Ala Ser 145 <210> 38 <211> 148 <212> PRT <213> artificial sequence <220><223><223>

Ala Val Thr Leu Asp Glu Ser Gly Gly Gly Leu Gin Thr Pro Gly Gly 10- 143761.doc 201125580 10 15

Gly Leu Ser Leu Val Cys Lys Ala Ser Gly Phe Ala Leu Thr Asp Tyr 20 25 30

Cys Val Gin Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Phe Val 35 40 45

Ala Ala lie Ser Asn Thr Gly Arg Tyr Thr Gly Tyr Gly Ser Ala Val 50 55 60

Lys Gly Arg Ala Thr lie Ser Arg Asp Asn Gly Gin Ser Thr Val Arg 65 70 75 80

Leu Gin Val Asn Asn Leu Arg Ala Glu Asp Met Gly Thr Tyr Phe Cys 85 90 95

Thr Arg Asp Gly Cys Ala Gly Cys Cys Gly Ser Tyr Tyr lie Asp Gly 100 105 110

Trp Gly His Gly Thr Glu Val lie Val Ser Ser Thr Ser Gly Gin Ala 115 120 125

Gly Gin His His His His His His His His His Gly Ala Tyr Pro Tyr Asp Val Pro 130 135 140

Asp Tyr Ala Ser 145 <210> 39 <211> 148 <212> PRT < 213 > 213 > artificial sequence <220><223> 223 > AH of VH of EnL4<400> 39 Ala Val Thr Leu Asp Glu Ser Gly Gly Gly Leu Gin Thr Pro Gly Gly 15 10 15

Ala Leu Ser Leu Val Cys Lys Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30

Tyr Met Phe Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45

Ala Gly lie Ser Gly Asp Gly Arg Tyr Thr Gly Tyr Gly Ala Ala Val 50 55 60

Asp Gly Arg Ala Thr lie Ser Arg Asp Asn Gly Gin Ser Thr Val Arg 65 70 75 80

Leu Gin Leu Asn Asn Leu Arg Ala Glu Asp Thr Gly lie Tyr Tyr Cys 85 90 95

Ala Arg Glu Ser Gly Ser Gly Cys Cys Asn Gly Asp Asn lie Asp Ala 100 105 110 c 143761.doc -11 - 201125580

Trp Gly His Gly Thr Glu Val He Val Ser Ser Thr Ser Gly Gin Ala 115 120 125

Gly Gin His His His His His His His His His Gly Ala Tyr Pro Tyr Asp Val Pro 130 135 140

Asp Tyr Ala Ser 145 <210> 40 <211> 150 <212> PRT < 213 > 213 > 221 > 221 < 223 > 223 > 223 > EnL5 VH of amino acid <400> 40

Ala Val Thr Leu Asp Glu Ser Gly Gly Gly Leu Gin Thr Pro Gly Gly 15 10 15

Ala Leu Ser Leu Val Cys Lys Gly Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30

Ala Met His Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45

Ala Gly lie Ser Arg Asp Gly Gly Ser Ser Thr Arg Tyr Tyr Gly Ala 50 55 60

Ala Val Lys Gly Arg Ala Thr lie Ser Arg Asp Asn Gly Gin Ser Ala 65 70 75 80

Met Arg Leu Gin Leu Asn Asn Leu Arg Ala Glu Asp Thr Ala Thr Tyr 85 90 95

Phe Cys Ala Lys Asp Ser Asp Asn Gly Gly Tyr Tyr Cys Asp Asp lie — _ 105 ' 110

Asp Ala Trp Gly His Gly Thr Glu Val lie Val Ser Ser Thr Ser Gly 115 120 125

Gin Ala Gly Gin His His His His His His His Gly Ala Tyr Pro Tyr Asp 130 135 140

Val Pro Asp Tyr Ala Ser 145 150 &lt;210&gt; 41 &lt;211&gt; 33 &lt;212> DNA &lt;213>Artificial Sequence&lt;220&gt;&lt;223&gt;Introduction Sequence&lt;400> 41 ggtggaattc tatctattct caagatccat gcc 12- 143761.doc 201125580 &lt;210&gt;&lt;211>&lt;212&gt;&lt;213&gt; 42 30 DNA artificial sequence &lt;220&gt;&lt;223&gt; inverted primer sequence &lt;40'0&gt; 42 actccatggt tacttggcca aggggtttct 30 &lt; 210> &lt;211> &lt;212> &lt;213> 43 39 DNA artificial sequence &lt;220&gt;&lt;223> pre-priming sequence &lt;400> 43 ccgcgtgaat tcggggatcc atgtctattc tcaagatcc 39 &lt;210> &lt;211&gt;&lt;;212>&lt;213&gt; 44 37 DNA artificial sequence &lt;220&gt;&lt;223&gt; Inverted primer sequence &lt;400> 44 catggagtcg acctcgagct tggccaaggg gtttctg 37 &lt;210> &lt;211> &lt;212> &lt;213> 45 7 PRT artificial sequence &lt;220&gt;&lt;223> synthetic sequence &lt;400&gt; 45 Gly Gly Ser Ser Arg Ser Ser 1 5 &lt;210> &lt;211&gt;&lt;212&gt;&lt;213&gt;&lt;220〉&lt;223> primer sequence &lt;400&gt; 46 ggssrssssg gggsgggg 18 &lt;210> &lt;211> &lt;212&gt;&lt;213> 47 24 DNA artificial sequence &lt;220> &lt;223&gt; primer sequence &lt;400&gt; 47 aagacagcta tcgcgattgc agtg 24 13- 143761.doc 201125580 &lt;210&gt; 48 &lt;211> 21 &lt;212> DNA &lt;213>Artificial sequence &lt;220&gt;&lt;223&gt;Introduction sequence &lt;4 (8) &gt; 48 ggtggttcct ctagatcttc c &lt;210> 49 &lt;211&gt; 104 &lt;212&gt; PRT &lt; 213 &gt; 213 &gt; artificial sequence&lt;220&gt;&lt;223&gt; </ br> genital VL &lt; 400 > 49

Ala Leu Thr Gin Pro Ser Ser Val Ser Ala Asn Pro Gly Gly Thr Val 15 10 15

Lys He Thr Cys Ser Gly Asp Ser Ser Tyr Tyr Gly Trp Tyr Gin Gin 20 25 30

Lys Ala Pro Gly Ser Ala Pro Val Thr Val lie Tyr Asp Asn Thr Asn 35 40 45

Arg Pro Ser Asn lie Pro Ser Arg Phe Ser Gly Ser Lys Ser Gly Ser 50 55 60

Thr Ala Thr Leu Thr lie Thr Gly Val Arg Ala Asp Asp Asn Ala Val 65 70 75 80

Tyr Tyr Cys Ala Ser Thr Asp Ser Ser Ser Thr Ala Gly lie Phe Gly 85 90 95

Ala Gly Thr Thr Leu Thr Val Leu 100 &lt;210> 50 &lt;211&gt; 142 &lt;212&gt; PRT &lt; 213 &gt; 213 &gt; artificial sequence&lt;220&gt;&lt;223&gt;&gt;223&gt; Amino acid sequence of germline VH &lt;400&gt; 50

Ala Val Thr Leu Asp Glu Ser Gly Gly Gly Leu Gin Thr Pro Gly Gly 15 10 15

Ala Leu Arg Leu Val Cys Lys Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30

Asp Met Leu Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Phe Val 35 40 45

Ala Gly lie Asp Asn Thr Gly Ser Tyr Thr His Tyr Gly Ala Ala Val 50 55 60 14- 143761.doc 201125580

Lys Gly Arg Ala Thr lie Ser Arg Asp Asn Gly Gin Ser Thr Gly Arg 65 70 75 80

Leu Gin Leu Asn Asn Leu Arg Ala Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95

Ala Lys Arg Thr Ala Gly Ser lie Asp Ala Trp Gly His Gly Thr Glu 100 105 110

Val lie Val Ser Ser Thr Ser Gly Gin Ala Gly Gin His His His His 115 120 125

His His Gly Ala Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ser 130 135 140 -15- 143761.doc

Claims (1)

201125580 VII. Scope of application: 1. An avian anti-α-isoolase I antibody, which specifically binds to α-isoenolase I. 2. The antibody of claim 1, wherein the bird is a chicken, duck or turkey. 3. The antibody of claim 1, wherein the bird is a chicken. 4. The multi-strain antibody of claim 1, which is a plurality of anti-α-isoenolase I IgY antibodies. 5. The multi-strain antibody of claim 1, which is an anti-α-isoenolase I chicken igY antibody. 6. A purified monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain immunoglobulin variable domain and a light chain immunoglobulin variable domain that binds to α-isoenolase I, wherein the light chain immunoglobulin The variable domain comprises the following amino acid sequence: (i) SGGSGSYG (SEQ ID NO: 1), SGGSSSYGYG (SEQ ID NO: 2), SGSSSGSYG (SEQ ID NO: 3), SGGSSSYGYS (SEQ ID NO: 4) in CDR1 Or SGSSGYGYG (SEQ ID NO: 5); (ii) ANTNRPS (SEQ ID NO: 6), NDNQRPS (SEQ ID NO: 7), RDDKRPS (SEQ ID NO: 8), SNNQRPS (SEQ ID NO: 9) or SNDDRPS (SEQ ID NO: 10); and (iii) GGYDSSAGI (SEQ ID NO: 11), GSGDSSTGM (SEQ ID NO: 12), GSGESSTNNGI (SEQ ID NO: 13), GSMDSSNSGV (SEQ ID) in CDR3 NO: 14) or GGYDSSASYVGI (SEQ ID NO: 15); and wherein the heavy chain immunoglobulin variable domain comprises the following amino acid sequence: (i) SFNMF in CDR1 (SEQ ID NO: 16) 'SHDMG (SEQ ID NO: 17), DYCVQ (SEQ ID NO: 18) 'SFYMF (SEQ ID NO: 19) or SYAMH (SEQ ID NO: [143761.doc 201125580 20); (ii) CDR2 GINNAGSTTNHGAAVKG (SEQ ID NO: 21), GIENAAGIGTFYGAAVKG (SEQ ID NO: 22), AISNT GRYTGYGSAVKG (SEQ ID NO: 23), GISGDGRYTGYGAAVDG (SEQ ID NO: 24) or GISRDGGSSTRYYGAAVKG (SEQ ID NO: 25); and (iii) SPGGIDGIDG (SEQ ID NO: 26), GADTGGWPAANIDA (SEQ ID NO: 27) in CDR3 ), DGCAGCCGSYYIDG (SEQ ID NO: 28), ESGSGCCNGDNIDA (SEQ ID NO: 29) or DSDNGGYYCDDIDA (SEQ ID NO: 30). 7. The purified monoclonal antibody or antigen-binding fragment thereof according to claim 6, which comprises a VH and VL FR1, FR2, FR3 and FR4 framework selected from the germline gene sequence (SEQ ID NO: 31) of chicken immunoglobulin. The framework region or amino acid sequence of the region is at least 85% identical to the framework regions of the VH and VL FR1, FR2 and FR3 framework regions encoded by the germline gene sequence of chicken immunoglobulin (SEQ ID NO: 31). 8. The purified monoclonal antibody or antigen-binding fragment thereof according to claim 7, which comprises an amino acid sequence selected from the VHs encoded by the germline gene sequence of chicken immunoglobulin (SEQ ID NO: 31) and At least 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 97%, 98% or 99% of the framework regions of the VL FR1, FR2 and FR3 framework regions. 9. The purified monoclonal antibody or antigen-binding fragment thereof according to claim 6, which comprises a VL complementarity determining region having an amino acid sequence selected from the group consisting of the following amino acid sequences: i) SEQ ID NO in CDR1: 1. SEQ ID NO: 6 in CDR2 and SEQ ID NO: 11 in CDR3; ii) SEQ 143761-doc in CDR1 201125580 ID NO: 2. SEQ ID NO: 7 in CDR2 and SEQ ID NO in CDR3 : 12 ; iii) SEQ ID NO: 3 in CDR1, SEQ ID NO: 8 in CDR2 and SEQ ID NO: 13 in CDR3; iv) SEQ ID * NO: 4 in CDR1, SEQ ID NO in CDR2 SEQ ID NO: 5 in SEQ ID NO: 14 and CDR3, SEQ ID NO: 5 in CDR2, SEQ ID NO: 10 in CDR2 and SEQ ID NO: 15 in CDR3. A purified monoclonal antibody or antigen-binding fragment thereof according to claim 6, which comprises a VL complementarity determining region having an amino acid sequence selected from the group consisting of the following amino acid sequences: ii) SEQ ID NO in CDR1 2, SEQ ID NO: 7 in CDR2 and SEQ ID NO: 12 in CDR3; and v) SEQ ID NO: 5 in CDR1, SEQ ID NO: 10 in CDR2 and SEQ ID NO: 15 in CDR3 . 11. The purified monoclonal antibody or antigen-binding fragment thereof according to claim 6, which comprises a VL complementarity determining region having an amino acid sequence selected from the group consisting of SEQ ID NOS: 3 1-35 and having a SEQ ID NO selected from the group consisting of SEQ ID NO : The &quot;V Η complementarity determining region of the amino acid sequence of the group consisting of 36-40. 12. The purified monoclonal antibody or antigen-binding fragment thereof according to claim 11, which comprises a VL^complementarity determining region having the amino acid sequence of SEQ ID NO: 32 or SEQ ID NO: 35 and having SEQ ID NO: 37 Or the VH complementarity determining region of the amino acid sequence of SEQ ID NO: 40. 13. A pharmaceutical composition comprising a polyclonal antibody of claim 1 or a monoclonal antibody of claim 6 and a pharmaceutically acceptable carrier. A method for treating or preventing an α-isoenolase I-related disorder, comprising administering a therapeutically effective amount of the polyclonal antibody of claim 1 or the antibody of claim 1 143761.doc 201125580 strain of claim 6. The method of Item 14 wherein the condition is selected from the group consisting of: an autoimmune disorder, ischemia, and a method of sipping a chain, wherein the cancer is selected from the group consisting of Cancer cancer, anal cancer, bladder cancer, bone cancer, intestinal cancer, brain cancer, leukemia, liver cancer, pancreatic cancer, prostate cancer, rectal cancer. 17. The method of claim 16, wherein the cancer is lung cancer. 18. The method of claim 17, wherein the lung cancer is non-small cell lung cancer. I% species test sample "• Heterogeneous enzymes! The presence of an in vitro diagnostic enol may comprise: (1) contacting the sample with an anti-α-isoenzyme I antibody as claimed in claim 1 or claim 6; and (ii) detecting a complex between the α_ samples The formation of objects. A 1 antibody and the 20: Γ2 detection kit for the presence of α-isoenolase 1 in the sample, 1 package of the anti-cardinal enolase of claim 1 or claim 6! Antibody or its fragments&apos; and informational materials as appropriate. 21. The kit of claim 20, and the antibody of the individual strain of 6ρ. The anti-isosinase 1 antibody is a set of antibodies as described in claim 22. Iso (tetra) enzyme 1 antibody is 143761.doc