MXPA98009430A - Essays to measure proteic fragments in media biologi - Google Patents

Abstract

The present invention relates to new antibodies and versions obtained by genetic engineering thereof, and a methodology for controlling protein fragments in a biological medium, especially fragments of collagen resulting from the cleavage of type II collagen by collagen

Description

TESTS TO MEASURE PROTEIN FRAGMENTS IN BIOLOGICAL MEDIA FIELD OF THE INVENTION This invention relates to methods for detecting protein fragments in a biological medium. More specifically »refers to procedures to quantify collagen fragments that result from the cleavage of type II collagen by collagenase.

BACKGROUND OF THE INVENTION The physiological renewal of articular cartilage represents a delicate balance between synthesis and degradation. It is a characteristic of normal growth and development and maintenance of cartilage in the adult. The net loss of cartilage is a characteristic of rheumatoid arthritis and osteoarthritis. This loss is very associated with incapacitation and with a low quality of life. Cartilage destruction in rheumatoid arthritis and osteoarthritis is currently diagnosed based on a combination of clinical symptoms and radiological findings. In the early stages of the disease, injuries to the articular cartilage occur long before they can be detected radiologically; lesions are detected radiologically only after extensive and probably irreversible cartilage losses have occurred. Therefore »it is critically important that physicians have biochemical markers for the early diagnosis of cartilage lesions» so that therapy can be initiated in the early stages »before extensive lesions occur. Type II collagen makes up the major part of the fibrillar skeleton of the cartilage matrix "just as type I collagen forms the fibrillar organization of the extracellular matrix of most other tissues" such as skin »bones» ligaments and tendons. These collagens are composed of a tightly bound triple helix that can only be cleaved by collagenase etaloproteinases, producing fragments of chain a with a length of 3/4 and 1/4 that are identifiable by electrophoresis in polyacrylamide gel. The destruction of articular cartilage during arthritic disease is due "in part" to the degradation of the tracelula matrix. which is composed mainly of type II fibril collagen and proteoglycans that are added. In articular cartilage, type II collagen fibrils are responsible for tensile strength »while proteoglycans provide the compression resistance needed for normal function and articulation. The precise mechanisms by which these connective tissue components are degraded are not fully understood. In mammals, "an important mechanism involves collagenases" which are a group of enzymes capable of site-specific cleavage of helical (native) collagen.

BRIEF DESCRIPTION OF THE INVENTION This invention comprises a method for controlling protein fragments, preferably fragments of collagen, in a biological medium, said fragments resulting from the cleavage of collagen type II by collagenase comprising; contacting said biological medium with a capture antibody; said capture antibody being active against the sequences indicated in the sequence listing as SEQ ID NOS: 1 and 2; and »in a second step» contacting said biological medium with a detection antibody »said detection antibody being active against the sequences indicated in the sequence listing as SEQ ID NO.

NOS: 3 and 4; and »finally» detecting the amount of collagen fragments attached to said capture and detection antibodies using conventional techniques that are well known to those of ordinary skill in the art. The specialists in this technique will recognize that the order of contact of the antibodies with the biological means can be reversed. Therefore "in another aspect" this invention comprises a method for controlling protein fragments in a biological medium »comprising; detecting the amount of collagen fragments bound to said capture and detection antibodies or; contacting said biological medium with a capture antibody; said active capture antibody being against the sequences indicated in the sequence listing as SEQ ID NOS: 3 and 4; and contacting said biological medium with a detection antibody, said detection antibody being active against the sequences indicated in the sequence listing as SEQ ID NOS: 1 and 2? and detecting the amount of collagen fragments attached to said capture and detection antibodies. In a preferred aspect, this invention provides a method for the detection of protein fragments which are fragments of collagen generated by the cleavage of the articular cartilage by collagenase and »more particularly» a method in which said protein fragments are generated from the cleavage of type II collagen by collagenase. In yet another aspect, this invention provides a third method for controlling collagen fragments generated from the articular cartilage in a biological environment comprising; contacting said biological medium with an active antibody shows the sequences indicated in the sequence listing as SEQ ID NOS: 3 and 4; and detecting the amount of collagen fragments bound to said antibody. In a broader aspect, this invention provides a method for controlling protein fragments in a biological medium, comprising; contacting said biological medium with an antibody capable of recognizing and binding protein fragments -which contain the sequences indicated in the sequence listing as SEQ ID NOS: 3 and 4; and detecting the amount of protein fragments bound to said antibody. This invention provides a capture antibody that is a monoclonal antibody. This invention also provides a detection antibody that is a monoclonal antibody. This invention provides a monoclonal antibody "designated 9A4" having the vH sequence indicated in the sequence listing as SEQ ID NO: 5 and the Vc sequence indicated in the sequence listing as SEQ ID NO: 6. This invention provides a antibody which is an antibody obtained by genetic engineering and having a similar sequence "but not necessarily identical to the scFv of p9A41CAT7-l (ATCC 9B593), ATCC, American Type Culture Collection» 10801 University BIvd. »Manassas» Virginia 20110-2209 USA , and p9A41F-5 (ATCC 98592), corresponding to SEQ ID NOS: 7 and 8 indicated in the sequence listing, respectively. This invention provides an antibody which is an antibody obtained by genetic engineering and having a sequence similar, but not necessarily the same as the scFv 5109 of p5109CscFv7 (ATCC 98594). indicated in the sequence listing as SEQ ID NO: 9. This invention provides a monoclonal antibody "designated 5109" having the sequence of ,, shown in the sequence listing as SEQ ID NO: 10 and the sequence of V ^ shown in the sequence listing as SEQ ID NO: 11. This invention provides a method for detecting protein fragments using an antibody »termed" 5109"and having the sequence of V, -. shown in the sequence listing as SEQ ID NO: 10 and the sequence of ^, shown in the sequence listing as SEQ ID NO: 11. In another aspect »this invention provides a hybridoma cell line that produces a monoclonal antibody that it binds to peptides that consist essentially of the structure indicated in the sequence list as SEQ ID NO: 1 or SEQ ID NO: 2, with the cell line having the ATCC identification characteristics HB-12436. In another aspect, this invention provides a hybridoma cell line that produces a monoclonal antibody that binds to peptides that consist essentially of the structure indicated in the sequence listing as SEQ ID NOS: 3 or 4 »having the cell line the identification characteristics of ATCC HB-12435. In yet another aspect, this invention provides a culture of E. coli that produces an antibody obtained by genetic engineering related to 9A4 »that binds to peptides that consist essentially of the structure indicated in the sequence listing as SEQ ID NOS: 1 or 2. In another aspect, this invention provides a culture of E. coli which produces an antibody obtained by 5109 related genetic engineering which binds to peptides consisting essentially of the structure indicated in the sequence listing as SEQ ID NOS: 3 or 4. In another further aspect »this invention provides a bispecific antibody produced by hybridization of antibodies 9A4 and 5109, where each half of antibody recognizes its respective binding molecule. In another aspect, this invention provides a bispecific antibody which is a genetically engineered combination of antibodies 9A4 and 5109 »produced by the combination of the V ^ and ^ domains of the two antibodies in the form V ^ r 5109) -link -VH (5109) -engar-VZ (9A4) -link-V? (9A4) and equivalents thereof. This invention further provides a method for controlling fragments of collagen in a biological medium, comprising: contacting said biological medium with a bispecific antibody 9A4 / 5109 previously described; and detest the amount of collagen fragments bound to said antibody. This invention also provides a bispecific antibody produced from genetically modified variants of antibodies 5109 and 9A4.

DESCRIPTION OF THE DRAWINGS Figure 1 is a standard curve of peptide 130 (SEQ ID NO: 20) and resultant optical density reading determined by Sandwich Elisa of Capture 9A4 / 5109-Bt; Figure 2 is a standard surva for the 5109 sandwich assay of detection 9A4-Bt; and Figure 3 is a standard curve of inhibition of the binding of 5109a by the pepOS4 (SEQ ID NO: 19) pending versus 054 concentration.

DETAILED DESCRIPTION OF THE INVENTION Type II collagen is the structural protein that provides the articular cartilage with its tensile strength and shear strength. It also provides the structural basis for containing the proteoglycan »which imparts the compressive strength and by doing this directly determines the osmotically pressurized cartilage shape. Thus, the structural integrity of type II collagen is an important determinant of the physical properties and durability of articular cartilage. The progressive deterioration of articular cartilage is one of the hallmarks of arthritic disease. When this deterioration is based on changes in the structure of type II collagen, it would be advantageous to have a methodology to specifically measure the decomposition of type II collagen. Fragments of type II collagen from the articular cartilage are released into the synovial fluid, in the lymph »in the blood and in the urine as the type II collagen is fragmented. The measurement of the surviving fragments could provide a procedure to control the decomposition of type II collagen in order to detect the onset of the arthritic disease and to measure the progression of the disease. In addition »it would also be useful to measure the effect of therapy on the decomposition of type II collagen during the disease. A variety of procedures have been used to control the decomposition of collagen. In mammalian tissues, it seems that collagenase is the extracellular enzyme limiting the speed involved in decomposing type II solágen (1). The ragmentation by collagen solagenase in three surtars and a one-quarter piece was already identified in 1967 (2,3) and there are currently three mammalian collagenases identified to be involved in the decomposition of type II solágen (4,5). There are other enzymes involved in the additional ragmentation of type I collagen II. The lysosomal decomposition of fibrillar collagens in bone and liver is known (6 * 7). Since collagen is one of the few proteins characterized by a high content of hydroxyproline, the measurement of hydroxyproline in urine has been examined as a measure of the turnover of collagen (B). However, as type I and type III collagens are found in large quantities in the skin, bone and connective tissues in general, the procedure has not been considered useful for measuring the breakdown of any particular type of collagen or collagen. 'of a particular compartment of the body' eg 'bone and has no value in controlling type II collagen' as it provides only an extremely small portion of the daily urine secretion of hydroxyproline (8). Therefore, efforts to control the breakdown of collagen or collagen itself have been based on immunological procedures. The antibodies can distinguish unique collagen fragments for a type of collagen and a cleavage site and potentially control the specific mode of collagen decomposition (9). Polyclonal and monoclonal antibodies against type I and III collagen or its fragments have been prepared; and tests have been prepared for the decomposition products of type I and type II collagens, for example »see Eyre (10). Relatively few methods have been presented to create antibodies against the decomposition products of type II collagen.

Polyclonal and monoclonal antibodies against type II solágen have been prepared (9, 11-13). These antibodies have been used for the detection of intact type II collagen rather than for the quantitative determination of collagen fragments. However »Eyre (14) has prepared monoclonal antibodies against fragments of type II collagen that contain the cross-linking residues and created an assay to determine the decomposition of type II collagen based on the cross-linking fragment that contained a similarly specific sequence for type II collagen similar to that indicated in its issued patent (10). Dodge and Poole prepared polyclonal antibodies against denatured type II collagen that were not reactive with other collagens (15, 16). The epitope was sequenced and later Hollander and Poole (17,18,19), prepared a competitive assay of antibodies against fragments of type II collagen that had the sequences indicated in the sequence list as SEQ ID NOS: 12 and 13 »Using a monoclonal antibody. Billinghurst et al. - (20) have prepared polyclonal antibodies against the neoepitope of the type II sol allegen cleavage site (which has the sesuensia indicated in the sequence listing as SEQ ID NO: 2) and have prepared a competitive assay of type II collagen. Srinivas, Barrach and Chishester (21-23) have prepared multiple monoslonal antibodies for a type II collagen assay using the sanogen bromide fragments of type II collagen as antigens. Although the reactive epitopes with the antibody have not been identified (24), these can also be used to test type II collagen. This invention provides two types of assays for type II collagen metabolism. The two assays are based on an antibody (polyclonal, monoclonal or genetically engineered antibody) against a defined sequence of type II collagen against which antibodies have not previously been prepared. The sequence is rich in acidic residues, ie the sequence indicated in the sequence list as SEQ ID NO: 3, in which a deletion of 1 residue has been made at the C-terminal end. Since no aspartyl or extracellular glutamyl endopeptidase has been described in mammals, collagen fragments rich in acidic residues would survive additional metabolism and could be measured in body fluids. Antibodies against these residues or against a fragment of collagen containing these residues would provide a general method of detecting type II fragments in body fluids, regardless of the collagen metabolite generation process. This invention provides monoclonal antibodies 5109 and variants obtained by genetic engineering, which are specific for the sequence of type II collagen and which bind to collagen fragments containing the sequence. The first trial is a general procedure to evaluate the discomposition of type II collagen. This assay provides a general competitive procedure for quantifying the amount of the sequence indicated in the sequence listing as SEQ ID NO: 3 »in which a deletion of 1 residue has been made at the C-terminus, and its closely related congeners . For the second assay, an additional antibody was prepared which is directed against the induction sequence in the list of sesoces as SEQ ID NO: 14. There is a C-terminal carboxyl group in the glycine (residue 9 of SEQ ID NO: 14) . This sequencing is obtained when the collagenase cleaves the type II solágen and, therefore, is classified as a neoepitope, that is, it is not present in the native sequence (the sequence indicated in the list of sequences as SEQ ID NO: 15 »Continues -GPOGPQG / LAG-, where the collagenase cleaves by the vertical bar) but appears when the collagen is cleaved by the solagenase. Previously, polyclonal antibodies against this sequence have been presented (21). Monoclonal antibody 9A4 and certain genetically engineered derivatives thereof react with the sequence of the neoepitope indicated in the sequence listing as SEQ ID NO: 2, but do not react with type II collagen or non-cleaved type I collagen. Although the sequence of the neoepitope is unique for collagenase, type I collagen generates a homologous sequence with a weak cross-reactivity when split by the solegenase (SEQ ID NO: 16) as indicated in the list. of sequences). This also occurs in the case of type III collagen; generates the sequence are weak cross-reactivity indicated in the list of sequens as SEQ ID NO: 2, when cleaved by collagenase. Thus, the neoepitope antibody lacks total speci? City for type II collagen and could not selectively detect cleavage of type II collagen by collagenase if used alone. However, when antibody 5109 and antibody 9A4 are combined in a sandwich assay, the two antibodies together can selectively detect the type II collagen metabolites generated by collagenase. Furthermore, an advantage provided by the test sandwich type of this invention is that an increase in sensitivity of 100 to 1000 times can be achieved with respect to a simple competitive assay based on 9A4 alone. Thus, the sandwich assay format with the antibodies described in this invention provides a unique method for controlling the metabolism of type II collagen by the solagenase under normal and pathological conditions, which has not been previously described. The 9A4 antibody is only a new monoclonal antibody that has utility for the detection of type I collagen fragments of type II or of type III »cleaved by collagenase» whenever it is not necessary to distinguish the type of collagen.

REFERENCES 1. Harris ED »Jr» Krane S. Collagenases. N Engl J Med 1974; 291: 557-563, 605-609, 652-661. 2. Nagai Y »Lapiere CM» Gross J. Tadpole collagenase. Preparation and puri isation. Bioshemistry 1966; 5: 3123-3130. 3. Sakai T, Gross J. Some properties of the production of reaction of tadpole collagenase with collagen. Biochemistry 1967; 6: 518-528. 4. Pendas AM »Matilla T» Estivill X »Lopez-Otin C. The human collagenase-3 (CLG3) gene is located on chromosome llq22.3 clustered to other members of the matrix metalloproteinase gene fa ily. Genomics 1995; 26: 615-8. 5. Mitchell PG, Magna HA, Reeves LM, Lopresti- Morrow LL »Yocum SA» Rosner PJ, Geoghegan KF, Hambor JE »Cloning * expression, and type II collagenolytic activity of matrix metalloproteinase-13 form human osteoarthritis cartilage, J clin Invest 1996; 97: 761-8. 6. Masiewisz RA, otton SF, Etherington DJ, Duance VC »Susceptibility of the cartilage collagens type II» IX and XI to degradation by the protein proteinases »cathepsins B and L» FEBS Lett 1990; 269: 189-193. 7. van Noorden CJF, Everts V. Selestive inhibition of cysteine proteinases by z-phe-alaCH-aF suppresses digestion of sollagen by fibroblasts and oeteoslasts. Biochem Biophys Res Comm 1991; 178: 178-184. 8. Kivirikko, K.S. Urinary secretion of hydroxy-proline in health and disease. Int. Rev. Connect Tissue Res. 1970; 5, 93-163. 9. Timpl R. Antibodies to collagens and prosollagen. Methods Enzymol 1982; 82: 472-498. 10. Eyre D. Patent. U.S. 5,320,970. 11. Holmdahl R »Andersson M» Tarkowski A. Origin of the autoreastive anti-type II collagen response »I, Frequency of specific and ultispecific B-cells in primed murine lymph nodes» Immunology 1987; 61: 369-374. 12. Punjabi CJ, Wood DD, Wooley PH. A monoclonal anti-type II sollagen antibody with cross reactive anti-lg activity specific for the F (ab ') a fragment. J Im unol 1983 141: 3819-3822. 13. Jasin HE »Taurog JD. Mechanisms of disruption of the articular cartilage surfase in inflammation. Neutrophil elastase increases availability of collagen II epitopes for binding with antibodies on the surface of articular cartilage. J Clin Invest 1991; 87: 1531-1536. 14. Orlund LL, Shao P, Yoshihara P, Eyre DR. Markers of bone type I and cartilage type II collagen degradation in the Hartley guinea pig model of osteoarthritis. Trans Orthoped Res Assoc 1997; 22: 313. 15. Dodge GR »Poole AR. Im unohistoshemisal detestion and immunochemícal analysis of type II collagen degradation in human normal »rheu atoid and osteoarthritic articular cartilages and in explants of bovine articular cartilage sultured with interleukin-1. J Clin Invest 1989; 83: 647-661. Iß. Dodge GR »Pidoux 1» Poole AR. The degradation of type II collagen in rheumatoid arthritis: an im one electron microscope study. Matrix 1991; 11: 330-338. 17. Poole AR. WO 94/14070. 18. Hollander AP, Heathfield TF, Webber C, Iwata Y »Bourne R, Rorabeak C. Poole AR, Insufficient damage to type II collagen in osteoarthritic articular cartilage detected by a new immunoassay» J Clin Invest 1994; 93: 1722-32. 19. Hollander AP, Pidoux I, Reiner A, Rorabeck C, Bourne R »Poole AR» Damage to type II collagen in aging and osteoarthritis starts at the articular surface »originates around chondroaytes, and extende into the sartilage with Progressive degeneration, J Clin Invest 1995; 96: 2859-69. 20. Billinghurst RC, Dahl? Serg L, lonessu M, Reiner A, Bourne R »Rorabeck C» Mitchell P, Ha bor J, Diekmann O, Tseshesche H, Chen J, van Wart H, Poole AR »Enhansed cleavage of type II collagen by collagenases in osteoarthritic articular cartilage. J Clin Invest 1997; 99: 1534-1545. 21. Srinivas GR »Barrach HJ» Chichester CO. Quantitative immunoassays for type II collagen and ite synogen bro ide peptides. J Im unol Meth 1993; 159: 53-62. 22. Srinivae GR »Chishe ter CO, Barrash HJ, Matoney AL. Effects of certain antiarthritic agents on the synthesis of type II collagen and glycosaminoglycans in rat shondrosarcoma cultures. Agent Actions 1994; 41: 193-199. 23. Srinivas GR, Chichester CO »Barrach HJ, Pillai V» Matoney AL. Production of type II collagen specific monoclonal antibodies »Immunol Invest 1994; 23: 85-98. 24. Chichester CO »Barrach HJ» Srinivae GR, Mitchell P. Immunological detection of type II collagen degradation: use in the evaluation of anti-arthritic therapies. Phar Pharmacol 1996; 4B: 694-69B.

DEFINITIONS Immunoglobulin (Ig): A natural tetrameric protein composed of two light chains of approximately 23 kD and two heavy chains of approximately 53-70 kD, depending on the amino acid sequence and the degree of glycosylation. Multimers of the tetrameric protein (IgM and IgA) are also formed. There are two classes of light chains, Kappa (k) and lambda (¡\) "and several kinds of heavy chains" gamma (), mu (μ), alpha (oc) »delta (< =) and epsilon (e) . There are also subclasses. Each chain, be it a light chain or a heavy chain, is made up of two parts. The first part, which begins at the N-terminus end of each chain, is called the variable domain. The C-terminal half of the light chain is called the constant region of the light chain and is the main determinant whether the light chain ee of type k or. The snozen region of the heavy chain comprises approximately the three fourths of the C-terminal heavy chain and determines the class of the immunoglobulin molecule (IgG-t, IgM »etc)» that is »a heavy chain X corresponds to an IgG and a μ heavy chain corresponds to an IgM * etc. - ^ y. VM: The amino acid sequence of the variable domain of each light chain (V ^ and the variable domain of the heavy chain (V ,,) together determine the binding specificity and the binding constant (kD) of the immunoglobulin molecule. variable domain comprises approximately half the length of the light chain and approximately one length of the heavy chain, and in the two chains it begins at the N-terminal end of the chain. (3) hypervariable segments known as complementarity determining regions or CDRs CDR and F: Each variable domain »V- ^ and VH» is comprised of three CDRs: CDR1 »CDR2 and CDR3 The segments interposed in the sequencing before and after Each of the CDRs is composed of four FR segments: FRl »FR2» FR3 and FR4, Vt. and V \: The Vt domain is ko? depending on which constant region (C- ^ or C is used during the productive rearrangement of the light chain (C- ^ or VJC ^ Antibody: Antibodies are specific immunoglobulin molecules produced by the B cells of the immune system in response to protein exposures, glycoproteins »viral cells, chemical compounds coupled to vehicles and other substances. An antibody is simply an immunoglobulin molecule for which its binding molecule is known. The substance to which the anti- body is attached is called an antigen. The binding of such antibodies to their antigens is highly refined and the multitude of specificities capable of being generated by sambioe in the amino acid sequence in the variable domains of the heavy and light chains is remarkable.

Polyslonal antibody: Normal immunization leads to a wide variety of antibodies against the same antigen. Although each B lymphocyte normally produces an immunoglobulin molecule with a defined amino acid sequence, many B lymphosites are stimulated in an immune response to make immunoglobulin molecules that react with the antigen, i.e., antibodies. These different antibodies are sarasterized by different amino acid molecules in the variable regions of the immunoglobulin molecule which result in differences in the fine specificity and affinity of the binding. Such antibodies are termed polyclonal antibodies to emphasize the diversity of binding specificities and binding constants that arise from the diversity of amino acid sequelae ensontradae in the different immunoglobulin molecules used in the immune response.

Monoslonal Antibody (MAb): A B lymphocyte that produces a single antibody molecule can hybridize with an immortal B cell lymph cell line "ie, a myeloma" to obtain an immortal antibody-producing cell line »ie, a hybridoma. The hybrids thus formed are segregated into individual genetic strains by selection, dilution, subcloning and restraint and, therefore, each strain represents a single genetic line. It produces an eolo antibody with a unique sequence. The antibody produced by such a cell line is called "monoclonal antibody" or MAb »with reference to its pure genetic lineage and differentiating itself from the polyclonal antibody» produced from mixed genetic ancestors »that is, multiple B cells. As a MAb ee a pure chemical reastive, it provides consistent and uniform results in immune assays. Furthermore, since MAB is produced by an immortal cell line, the reagent euminietro is not limiting. For these reasons, a MAb is much more preferred than polyclonal antibodies for diagnostic purposes. Antibody obtained through genetic engineering: As the binding tradition of a reeide antibody in the variable regions of the light and heavy chains, the antibodies can be engineered to change or eliminate the cont regions and, if this is done properly, can produce an antibody molecule with different properties and molecular weight »but with equal or very similar antigen binding properties. For example, the V ^ and V- ^ genes can be cloned and assembled (or VH and V.;.) With an appropriate linker between them. Such a new genetically engineered molecule is called a single chain antibody (abbreviated as scFv) and typically has a molecular weight of 25 to 28 kD »depending on the design of the linker and the addition of other sequences to aid purification, stability , circulation »detection» etc. Multimers of the single-chain antibody can also be obtained by the appropriate use of linkers in which the order of each pair V ^ can vary, and v. In addition »some changes in the amino acid sequencing of the V ^ and? Regions can be made. that retain the desirable antigen binding properties. It can be seen that from the original sequence of an antibody "an infinite diversity of engineered antibodies can be obtained which retain the specificity of antigen binding" but which are created to meet certain specific requirements. Other examples of antibodies obtained by genetic engineering include, but are not limited to, Fab »F (ab ') 2, chimeric antibodies, humanized antibodies, etc. For a review, see Winter G and Milstein C, "Man-made Antibodies", Natures 1991; 349, 243-299. Bispecific Antibody: Normally, an IgG antibody has two light chains and two identical heavy chains. Therefore, there are two identical antibody binding sites in the immunoglobulin molecule. On the contrary, a bispecific antibody is a single immunoglobulin molecule having two speci fi cities. It can be obtained by the fusion of two hybridoma cell lines producing monoclonal antibodies "where each hybridoma has a different antigenic specificity" and the selection of a cell line (a quadrone) that produces an anti-body whose somposission is a tetramer composed of a light chain and a heavy chain that come from each fused molecule of the hybridoma. The antibody produced by the quadroma has only one light chain and one peedal chain of each parenteral specificity, has a binding site for heavy / light chain sada and is bispecific, since it has two binding sites of different species. A bi-peptide antibody can also be obtained by genetic engineering. It may comprise the fragment V ^, - linker-V--, of an antibody linked by an additional linker to a V-.-- linker-Vj .. fragment of another anti-body molecule. The o of VL and V? it can be altered, but the final result is a bispecific anti- body.

Epitope: Depending on the size »structure and conformation of the antigen» an antibody can bind only a small part of the entire structure. The part of the antigen molecule to which the antibody binds is called its epitope. Different antibodies can be smeared for different epitopes on the same antigen.

Neoepitope: The antigen can have an epitope that is hidden »so that it can not bind to a specific antibody. However, a conformational change in the antigen may cause the appearance of the pro-deployment epitope or discovery of part of the superfisie of the molecule. This new state allows the antibody to bind to the epitope. In another aspect, the action of an enzyme on the antigen can cause the appearance of a new epitope to which the antibody can bind. For example, after excision by a proteolytic enzyme, new N-terminal and C-erminal sequences are generated. As the epitope is not observed in the parent molecule and as »after some change in the parent molecule, the epitope is revealed and can now bind to the antibody, it is called a neoepitope.

Biological medium: This can be defined as any biological fluid that could contain the antigen and be of interest to perform tests using this procedure. These include: blood, synovial fluid »urine. fluid, spinal »bronchiolar lavage fluid, linga, vitreous humor of the eye, tissue extracts, tissue culture supernatants» cartilage extracts »etc. It is not necessary to limit the biological medium to human samples, but it can also be obtained from a similar diversity of animal media (tests have been performed on mouse, rat, hamster, guinea pig, dog and cattle) in a similar way to the previous examples.

Immunoassays: An enemae of a substance (a biological complex such as a protein or chemical compound eimple) based on the use of the binding property of an antibody to recognize the substance that can be a specific molecule or a series of homologous molecules. The assay may include one or more antibodies.

Digestive assay: The antiserum binds directly to an antigen, such as an antigen present in a biological sample (cells, tissues »histological section» etc) or to an antigen adsorbed or chemically coupled to a solid surface. The antibody itself is typically labeled to allow determination of the amount of antibody bound to the antigen. Alternatively, the antibody (now called primary antibody) is detected with a marsed secondary antibody that will demonstrate that the binding of the primary antibody has occurred.

Competitive assay: An assay based on the binding properties of a single antibody molecule. Typically a labeled antigen that competes with an unknown antigen is used and the amount of unknown antigen is determined in terms of how much labeled antigen is displaced by the unknown antigen. The label can be radioactive, optical »fluorescent polarizing enzyme» fluorescent »fluorescent inastiator or another marker. The antibody can be onospecific or bispecific.

Sandwich Assay: This is a double antibody assay in which the two antibodies bind to the antigen "forming a trimeric or sandwish immune complex that is both antibodies with the antigen between them. An antibody is used to locate the immune complex on the surface or detection chamber. This antibody is called a capture antibody. The other antibody carries a marker that allows detecting the immune complex. It is called a detection antibody. If an immune complex is not formed (the antigen is not present) then the sapure antiserum can not carry the antithesis antibody to the debris. If the antigen is present, then an immune complex will be formed and the capture antibody will bind to the detection antibody so that the amount of detection antibody in the immune complex is quantitatively related to the amount of antigen present. The essay can be formatted in many ways. For example, the capture antibody can be chemically coupled to a solid surface, adsorbed in a nonspecific manner on a surface, bound by biotinylation to an avidin-like molecule, eg, avidin, streptavidin, neutravidin, etc., or a coated surface. with streptavidin or avidin »or coupling to magnetic particles or beads as a means of locating the immune complex in the measuring device. The detest antibody may be radioactive or may have a variety of possible enzyme amplification systems such as horseradish peroxidase (HRP), alkaline phosphataea (AP), urease, etc., when formatted as an ELISA (Immune Assay). with United Enzyme). It can have an electrochemical, optical, fluorescent or other detection method to determine the amount of detection antibody in the immune complexes. It can be seen immediately that many examples can be obtained in which the two antibodies are paired in a sandwich assay using a variety of methods to capture the immune complex in a detection device and a variety of detection systems to measure the amount of the immune complex.

Molecular biology techniques: As the present document provides the nucleotide sequences of the VH and V- ^ coding regions for the antibodies of the present invention, a person skilled in the art could produce in vitro a complete gene coding for the V regions? and V- ^ and a fully functional antibody. This can be produced as an immunoglobulin molecule of any given kind by adding constant regions of the light chain and the heavy chain or it can be produced as a scFv by joining V ^ and V ^ via a linker and adding marker fragments where appropriate. The constructed gene can be obtained by genetic engineering by conventional recombinant techniques, for example, to provide a genome insert in a plasmid capable of expression. Subsequently »the plasmids can be expressed in host cells» where the host cells can be bacteria »such as E. coli or a species of Basillus, yeast cells such as Pichia pastoris, or mammalian cell lines such as Sp2 / 0 cells, AgB or CHO.

Abbreviations: When the nucleic acids, amino acids, peptides, protecting groups, active groups and similar radicals are abbreviated, the abbreviation is used according to the IUPACIUB (Commission on the Biological Nomenclature) or the practice in the relased sampras. Following are examples.

Hybrid abbreviations HPLC High-pressure liquid chromatography SDS-PAGE Electrophoresis in polyacrylamide gel with sodium dodecyl sulfate PCR Chain reaction with Oligo polymerase Oligonucleotide TA Ambient temperature, approximately 22 ° C Reagents: EDTA Ethylenediamine tetraacetic acid SDS Dodecylsulphate sodium TW-20 Tween-20 NFDM Skimmed milk powder DPBS Phosphate buffered saline, Dulbecco Bt Biotinylated HAT Medium containing hypoxanthine »aminopterin and thymidine HT Medium containing hypoxanthine and HRP thymidine Horseradish peroxidase Molecules or chains similar to immunoglobulins V? Variable region of the heavy chain Vg, Variable region of the light chain scFv Single-chain antibody containing r. Y ? Nucleic Acids RNA Ribonucleic acid DNA Deoxyribonucleic acid cDNA complementary DNA mRNA messenger RNA Bases of nucleic acids Single-letter amino acid codes: Three-letter codes: Complete names. gly: glycine V: val: valine: leu: leucine ala: alanine I: ile: isoleucine S: ser: aspin: aspartic acid K: lis: lysine: arg: arginine his: histidine F: phe: phenylalanine Y: tyr: tyrosine thr: threonine C: cys: cysteine M: met: methyonin glu: glutamic acid W: -rp: tryptophan P: pro: proline hyp: hydroxyproline N: aspargin Q: gln: glutamine EXAMPLE 1 Generation and characterization of monoclonal antibody 9A4 Balb / c mice (Jackson Laboratories »Bar Harbor» ME) were immunized initially with the peptide having the sequence indicated in the Sequence Listing as SEQ ID NO. 17 (Anaspec »San JOE» CA) covalently bound to the maleimide KLH (Pierce Chemical, Rockford, ID »administered in Freund's complete adjuvant (DIFCO Detroit, MI) .The mice were subjected monthly to booster doses, for approximately 5 months »Using Freund's incomplete adjuvant (DIFCO» Detroit, MI), until the consentrations were 1: 100,000.The mice were subjected to iv reinforcement 10 days before fusion, splenosites were resolouted and fused were a sellar line that did not sesretaba Ig 'prosedente of P3X63Ag8.653 (Amerisa Type Culture Colletion ATCC, Bethesda, MD) using PEG-1450 at 505I (ATCC) were plated at a consension of 10ß cells / cell in 96-well microtiterplates * medium HAT (Sigma »St. Louis» MO) with fetal calf serum at 15JÍ (Hyclone, Provo »Utah) Ten days later» a selection of the posillos was made with a primary ELISA for the identification of positive posillos oe that produced antibodies, 10 ng / ml of biotinylated peptide (Bt-AEGPPGPQG) was added to streptavidin-coated plates (10 μg / ml) (Pierce Che isal) and 2 μl of hybridoma supernatant sada was added to 100 μl of DPBS (Gibso, Grand Island »NY) with TW-20 at 0.055Í (Sigma). Positive Eliea cells were detected by rabbit anti-mouse IgG-HRP (Jackson I muno Research, West Grove, PA). Eliea positive positios underwent a second round of selection in the BIAcore. We searched for the wells that produced antibodies and that had slow inactivation rates in the BIAcore "as determined using the software BIAevaluation version 2.1 (Pharmacia Biosensor, Piscataway» NJ). Using the Pharmacia Amine Coupling Kit (Pharmacia Biosensor) was conjugated streptavidin (Pierce * Chemical) »at a concentration of 100 μg / ml» with biosensor microplates coated with carboxylated dextran (Pharmacia Biosensor) at pH 4, O, using a flow rate of 5 μl / inuto for 35 minutes. Typically, 2000 RU were added. The biotenylated peptide (100 ng / ml) in residue 1 of SEQ ID NO: 14, as indicated in the Sequence Listing »was run on the microplate with estraptavidin at a flow rate of 100 μl / inute for 10 seconds. The candidate supernatants containing antibodies were stopped on the microplate (2 μl / min for 30 seconds) and the amount of antibody added was noted. The buffer was changed to HBS (Pharmacia Biosensor) and the rate of deociation was noted during the following 80 seconds. The misroplaque was cleaned with 0.1 N HCl for 30 seconds between each assay to remove residual antibody and to remove all nonspecific binding. Inactivation rates were determined using the BIAevaluation syn- thetic analysis software »version 2.1. The clones were selected to be the lowest inactivation rates for the subsequent analysis. These clones included 9A4, 11F2 and 3H10. An additional characterization of these clones was carried out as indicated in the following: each of preparasione sampled which were type I solágen, type I sol-es engendered by solagenaea »type II solágen and type II collagen exuded by collagenase, were coupled to a flow cell other than a BIA 2000 instrument. The Pharmacia Amine Coupling Kit (Pharmacia Biosensor) was conjugated to biosensor misroplaquetas coated with carboxylated dextran (Pharmacia Biosensor) at pH 4.0, using a flow rate of 5 μl / minute for 35 minutes. The four flow cells added SOOO »7000» 4000 and 4O00 RU respectively. The cells were washed are 0.1 N HCl between the tests for cleaners of all the uncoupled material and to clean them of all residual antibodies. All the antibody preparations were purified by chromatography are G protein (Pharmacia Biothecnology »Piscataway» NJ) and tested at 10 μg / ml. The total union was recorded to each of the four surfaces. The 9A4 antibody was selected because it showed selective binding to the type I soltanum emitted by the collagenase (type I = 11 RU; = 280 RU) and lacked significant binding to non-released collagen (type I = 3 RU, type II = 6 RU). After three rounds of subcloning by limiting dilution in HT medium (Sigma) are fetal calf serum at 554 (Hyalone) "a stable 9A4 onoslonal hybridoma was obtained. This has been deposited with the Americam Type Culture Collection as ATCC - HB - 12436.

EXAMPLE 2 Generation and Characterization of the 51Q9 Monoclonal Antibody The Balb / c mice were immunized initially with the peptide having the sequence indicated in the Sequence Listing as SEQ ID NO: 18 (Anaspec, San Jose »CA) covalently bound to the maleimide KLH (Pierce Chemical) and administered in adjuvant complete by Freud (DIFCO). Mice were subjected to monthly »reheating for about 5 months, using incomplete Freud's adjuvant (DIFCO) until the concentrations were 1: 100,000. The mice were reinforced i.v. IO days before the merger. Splenocytes were harvested and fused with a non-secreting cell line? G from P3X63Ag8.653 cells (ATCC) using PEG-1450 at 50? (ATCC). They were plated at concentrations of 10β cells / well, in 96-well microtiter plates, in HAT medium (Sigma) with fetal calf serum at 15 > í (Hyclone). Ten days later »the wells were selected through Elisa. For the identification of the positive progenitors of antibodies, 10 ng / ml of biotinylated peptide (biotinylated in residue 1 of SEQ ID NO: 19 as indicated in the Sequence List) were added to streptavidin-coated plates (10 μl). / ml) (Pierse Chemical) and 2 μl of each hybridoma supernatant was added to 100 μl of DPBS with TW-20 to O.OSíí (Sigma). Positive posillos del Elisa were detected by rabbit anti-mouse IgG-HRP (Jackson I munoResearsh). Positive posillos underwent a second round of selection in the BIAcore to detect those that had antibodies that gave the lowest rates of inactivation in the BIAcore. Streptavidin (Pierce Chemical) was conjugated at 100 μg / ml using the Pharmacia Amine Coupling Kit (Pharmacia Biosensor) to carboxylated dextran-coated biosensor micro-bottles (Pharmacia Bioseneor) at pH 4, using a flow rate of 5 μl / minute for 35 minutes. Typically, 2.OOO RU was added. The biotinylated peptide (10O ng / ml) was passed in the remainder 1 of SEQ ID NO: 19 »as indicated in the Sequence Layer, on the streptavidin chip at a flow rate of 100 μl / minute for 10 seconds. The supernatants that were antibody were passed over the myplate (2 μl / minute for 30 seconds) and the amount of antibody added was noted. The buffer was changed to HBS (Pharmacia Biosensor) and the dissociation rate was noted for the next 80 seconds. The microplate was cleaned with 0.1 N HCl for 30 seconds between each test to remove the antibody and to remove all non-specific binding. The inactivation rates were determined using the BIAevaluation software version 2.1. The clones were searched with slow inactivation rates. MAb 5109 was selected for further analysis. Each of four preparations consisting of collagen type I collagen type I collagenase - collagen type II and collagen type II - collagenase - were coupled to a single channel of a four channel Blacore. They were conjugated using the Pharmacia Amine Coupling Kit (Pharmacia Biosensor) to biosensing microplates coated with carboxylated dextran (Pharmacia Biosensor) at pH 4 »O» using a flow rate of 5 μl / minute for 35 minutes. Lae four flow cells added B000 »7000, 4000 and 4000 RU respectively. The cells were washed with 0.1 N HCl between the tests to clean them of all the non-coupled material and to clean them of all non-specific material. All antibody preparations were purified by protein G chromatography (Pharmacia Biotech) and assayed at 10 μg / ml.

The total union was registered to each of the smooth surfaces. Antibody 5109 was selected because it showed a selective binding to the collagen emitted by the collagenase (Type I extended = 23 RU, type II cleaved = 173 RU) but lacked signi icant binding to non-cleaved collagen (type I) = 23 RU; type II = 15 RU). After nine turns of subcloning by limiting dilution in HT medium (Sigma) -with fetal calf serum at% (Hyclone) »a stable monoslonal 5109 hybridoma was obtained. It has been deposited with the American Type Culture Collection as ATCC - HB - 12435.

EXAMPLE 3 Description of a sandwich assay using 9A4 as capture antibody and monoclonal 51Q9 as detection antibody Monoclonal antibody 9A4 (capture antibody) was added to SG well plates Nunc Maxisorp (VWR, Boston » MA) »being the antibody) 9A4 at a concentration of 10 μg / ml in borate buffer 0.05 M» pH 8.5 »and using 100 μl / well (except for the control wells with the numbers 4» 5 and 6, see Table 1) and incubated for 18- 48 hours at 4 ° C. The plate was washed three times with DPBS with TW-20 at 0. 05% (Sigma) »(DPBS / TW-20) and 200 μl / well was used. Wells of the plate blocked were 1% skimmed milk powder (NFDM) dissolved in freshly prepared DPBS (NFDMDPBS) * that is, kept on ice from no before the day of use »using 100 μl / well and incubating for 1 time at TA. The blocking solution was discarded and the wells rinsed once with 2O0 μl / DPBS / TW-20. The l30 peptide was diluted in 0.15 I NFDM DPBS at the concentrations shown in Table 1. Peptide 130 has the sequence in the Sequence Listing as SEQ ID NO: 20 and was synthesized and pued by Anaspec Inc. (San José »CA ). The results of the peptide 130 (SEQ ID NO: 20), the samples at the appropriate dilutions and the controls were collated in the specified posilloe of the misovaluation plate as shown in Table 1.

TABLE 1 Results of the misrovalorasion plate and antibody coating scheme pep130 (SEQ ID NO: 20) ng / ml TABLE 2 Additions to the control wells Controls: 5109 Biotinylated anti-biotin antibody 9A4 130 labeled with HRP 1 + - - + 2 + + - + 3 + - + + 4 - + + + 5 - + - + 6 - - + + The wells were washed three times with 200 μl / well of DPBS / T -20. 5109 monoclonal antibody conjugated with biotin '(5109-Bt) was added to all wells containing peptide 130 (SEQ ID NO: 20), to all sample wells and to all control wells except 1 and 2 and 5. Was 100 μl / well of 5109-Bt added at a concentration of 1 μg / ml * in NFDM DPBS to Oi? to each well * and the plate was incubated for 40 minutes at 37 ° C. Note: MAb was biotinylated using 37 μg of biotin-N-hydroxyceuccin ida (Pierce Chemical) per mg of MAb 5109 for 2 hours "and then dialyzed overnight using a dialysis cassette with a limit of 10 kD (Pierce Chemical ). The wells were washed three times with 200 μl / well of DPBS T -20. The anti-biotin mouse monoclonal antibody conjugated with HRP (Jackson I mnoResearch) was diluted 1/5000 in NFDM DPBS at 0.15% was added 100 μl / well to all wells and incubated for 30 minutes at RT. The posillos were washed three times are 200 μl / well of DPBS T -20. 100 μl / 1-step Turbo well (3.3 ', 55r-tetramethyl benzidine ready for use, Pierce Chemical) was added and incubated at RT for approximately 10 minutes. The color development was interrupted with HsSO ^ 2 N. The results were read on a spectrophotometer at 450 nm.

TABLE 3 Data of the standard curve for the sandwich assay of capture 9A4 / 5109 detection From the concentrations of peptide 130 and the resultant optical density readings at 430 nm (Table 3), a standard curve was constructed (Figure 1). Other appropriate peptides or fragments of collagen may be replaced to prepare a standard curve analogous to that of Figure 1. Units were expressed in terms of molar equivalents of the standard. In this case, the units were nM equivalents of peptide 130 (SEQ ID NO: 20). On the linear portion of the curve, a regression line was used to adjust the data. In the given case »the standard curve was linear between 0.735 nM and 0 * 46 nM when the concentrations are given in the logarithmic scale (as in the example, figure 1) and the regression curve is obtained using the linear portion of the curve. In the case of samples that fall outside the linear portion, the concentrations outside the graph can be read, the samples can be diluted so that they fall within the portion of the standard curve or they can be below the limit of detection. The regression between the log (nM) and the D0450 gives a slope of 0.029 D0450 / log (nM) and an ordinate at the origin of 0.024 D0450. When assaying unknown samples, the salivation surva can be used to determine the concentration of type II collagen fragments generated by the collagenase from the optical density of the sample. The following equation can be used: Log (concentration) = (Sample D0450 - Ordered in the Origin) / Pending Sample: In the given case, an unknown sample of synovial fluid had a D0450 of 0.229. Thus »Log (Consension) = (Sample D0450 - O, 024) / O, 029 =, 949 Taking the antilogarithm, the consentration of the fragment in the synovial fluid = 0,112 nM.

EXAMPLE 4 Description of a sandwich assay using monoclonal antibody 5109 somo capture antibody and 9A4 as detection antibody Monoclonal antibody 5109 (the capture antibody) was added to 9S Nunc Maxisorp well plates (VWR, Boston »MA) with antibody 5109 at a concentration of 10 μg / ml in 0.05 M sodium borate buffer pH 8.5 using 100 μl / well (except for control wells with numbers 4 »5 and 6, see Table 4) and incubated during IB-4B hours at 4 ° C. The plate was washed three times with DPBS with 200 μl / well of DPBS TW-20. The wells of the plate were blocked with 1% (NFDM) dissolved in freshly prepared DPBS »using 100 μl / well and incubated for 1 hour at room temperature. The blocking solution was discarded and the wells were rinsed once with 200 μl of DPBS / TW-20. Peptide 130 (SEQ ID NO: 20) was diluted in NFDM DPBS to Q. l'A to lae consentrasiones shown in table 4. Peptide 13? has the sequence indicated in the Sequence List as SEQ ID NO: 20 and was synthesized and purified by Anaspec Inc. (San José »CA). Dilutions of peptide 130 (SEQ ID NO: 20), unknown samples at the appropriate dilutions and controls were placed in the specified wells of the microtiter plate as shown in Table 4.

TABLE 4 Lacquer Results of Nickification and Antibody Coating Scheme pep130 (SEQ ID NO: 20) ng / ml TABLE 5 Additions to control wells Controls: 9A4 Biotin anti-biotin 5109 130 biotinylated labeled with HRP 1 + - + 2 + + + 3 + - + + 4 - + + + 5 - + + 6 - + The wells were washed three times with 200 μl / well of DPBS / TW-20. Monoclonal antibody 9A4 conjugated with biotin (9A4-Bt) was added to all wells containing peptide 130 (SEQ ID NO: 20), to all sample wells and to all control wells except 1, 2 and 5. 1O0 μl / well of 9A4-Bt was added at a concentration of 1 μg / ml in NFDM DPBS to the O.J.sub.y to the posillo and the plate was incubated for 40 minutes at 37 ° C. Note: 9A4 was biotinylated using 37 μg of biotin-N-hydroKisuccinamide (Pierce Chemical) per mg of monoclonal antibody 9A4 for 2 hours and then dialyzed overnight using a dialysis cassette with a limit of l? KD (Pierce Chemical). The wells were washed three times with 200 μl / well of DPBS TW-20. Anti-biotin mouse monoclonal antibody conjugated with HRP (Jackson I munoResearch) was diluted 1: 500O in NFDM DPBS to 0.15 I, and 100 μl / well was added to all wells and incubated for 30 minutes at RT. The wells were washed three times with 200 μl / well of DPBS TW-20. One hundred icoliters / 1-step Turbo well (3.3 *, 5,5'-tetramethyl benzidine ready for use, Pierce Chemical) was added and incubated at RT for about 10 minutes. The color development was interrupted is 2 N. The results were read on a spectrophotometer at 450 nm.

TABLE 6 Standard curve data for sandwich assay 5109 capture / 5109 detection From the sonsentrasionee of the peptide 130 (SEC ID NO: JO) the resulting optical density lesions at 450 nm, a standard curve was constructed. Again, other suitable peptides or collagen fragments can be used to prepare a standard curve. The units needed to be expressed in terms of pattern equivalents. In this case, the units were appropriate in terms of nanomolar equivalents of peptide 130 (SEQ ID NO: 20). On the linear portion of the surva, a regression line was used to adjust the data. In the given case, the standard curve was linear between 0.3125 nM and 0.0195 nM when the concentrations were given in the logarithmic scale (as in the example), figure) and the regression curve was obtained using the linear portion of the curve. For samples that fall outside the linear portion, the concentrations outside the graph can be read, the samples can be diluted so that they fall within the standard portion of the curve, or the concentration of the collagen fragments in the samples can be below the detection limit. The regression between the log (nM) and the D0450 gives a slope of O.42 D04SO / log (nM) and an ordinate at the origin of 0.70 D0450. When testing the samples, the calibration curve can be used to determine the concentration of the collagen fragments from the optical density of the sample.

Sample: In the given case, the unknown sample of human urine from an arthritic patient has a D0450 of 0.124. Log (concentration) = (Sample D0450 - 0.70) /0.42 = 1.3S To the antilogarithm, the concentration of the fragment in urine = 0.44 nM. In another case, the standard curve gave a slope of 0.249 and an ordinate at the origin of 0.638. An unknown sample of human plasma from a patient with osteoarthritis had a D0450 nm of 0.172. The osteoarthritic flap sample had a concentration of 68 pM. 4B EXAMPLE 5 The 51Q9 antibody can be used directly to measure the sanctities of type II collagen fragment in a competition assay In an adaptation of the concentration analysis (BIAapplications HandbooK, Pharmacia Bioseneor, June, 1994 Edition, pp. 6-2 to 6-9), streptavidin (Pierse Chemisal, Roskford »ID at a concentration of 100 μg / ml was conjugated to the Pharmacia Amine Coupling Kit (Pharmacia Biosensor) with carboxylated dextran-coated biosensor microplates (Pharmacia Biosensor) at pH 4.0, using a flow rate of 5 μl / minute for 35 minutes Typically, 2000 RU was added The biotinylated peptide was passed (100 ng / ml) having the sequence identified in the Sequence List as SEQ ID NO: 19, on the streptavidin chip at a flow rate of 5 μl / minute for 2 minutes, 144 RU of the peptide was added to the streptavidin surface. passed over the surface of the peptide, for one minute, at a flow rate of 10 μl / min, MAb 5109 at a concentration of 6.3 μg / ml either alone or in mixture with standard consenstrations of peptide 054 (SEQ ID NO: 19), or mixtures of 5109 and di tions of samples with unknown amounts of collagen fragments. The slopes of the linear portions of the association phase for each curve were analyzed with the BIAevaluation software version 2.1. A standard curve of the competition of peptide 054 (SEQ ID NO: 19) was constructed against the slope. The amount of collagen epitope in the samples was determined by comparing the slope of a sample with the slopes of the standard curve to calculate the amount of epitope. Between the injection, the chip was wiped with 0.1 N HCl for 30 seconds to remove the antibody.

TABLE 7 51Q9 Mixed with standard amounts of peptide 054 (SEQ ID NO: 19) Dil. of the values pep054 rO de regr. M isg M linear slope 1 1.34E-06 -5.87 0.187 2 6.71 E-07 -6.17 0.208 3 3.36E-07 -6.47 0.375 4 1.68E-07 -6.78 0.769 2 , 16 5 8,39E-08 -7,08 12,7 10,09 6 4,19E-08 -7,38 17,1 18,03 7 2,10E-08 -7,68 25,7 25,96 8 1.05E-08 -7.98 22.5 9 5.24E-09 -8.28 26.6 10 2.62E-09 -8.58 24.0 11 1.31 E-09 -8.88 26.6 Slope of linear regression = -26.36 ordered at the origin y = -176 Sample: Supernatant of an MMP13 digestion product of lagenase-3 solegenase of bovine nasal cartilage. The sample was tested on the BIAsore misroplate diluted 2 times, 4 times and B times. The calculated amounts of collagen were determined in terms of peptide 054 (SEQ ID NO: 19). The results are given in column 4 of sub-table 8. After multiplying by the titration, a molar concentration (M > of solágen in terms of the pattern O.54 (SEQ ID NO: 19) can be determined.

TABLE 8 The titration and concentrations of the unknown sample in terms of the concentration of peptide 054 (SEQ ID NO: 19) I concentrate pending M (054) nM (054) | 5109 only 6,648 Fund 5109 + 1: 2 3,539 4E-08 80 5109 + 1: 4 4,885 2E-08 80 5109 + 1: 8 5,825 8E-09 64 The consistency of the results (last column) after correction for dilution is demonstrated by the consistency of the calculated values from the three separate dilutions (following the last column) of the unknown samples. For the supernatant of bovine naeal cartilage, an average value of collagen fragments of type 4 75 nM is obtained.

EXAMPLE 6 Preparation of antibodies related to 9A4 by genetic engineering The basis for generating the antibodies by genetic engineering and their subsequent evaluation as biologically active or relevant molecules is the cloning, configuration of assembly and characterization of the Vt, and V? Domains. of the parental antibody. The structural sequences of V ^ and VH of the subject antibodies and the uniqueness of the partial combination v ^ -V ^ have been determined. which constitute the active binding site to the antigens described in this invention. Before cloning the genes of the 9A4 variable region, it was necessary to determine the protein sequence of portions of the variable domains of the parental IgGl 9A4 antigen, so that the variable domains would be cloned, it could be ascertained whether the variable domains had indeed been obtained sorrects and not other derivatives of the fusion partner of the myeloma or an inactive pseudogene from the B cell used in the generation of the hybridoma. A culture supernatant containing IgGl 9A4 was generated by developing the hybridoma 9A4 in cylindrical bottles. The supernatants were adjusted to pH 7.5 with dibasic sodium phosphate and the salt concentration was adjusted with 3 M sodium chloride to a final concentration of 150 mM. The filtered supernatant (0.2 μ) was passed through a bed volume of 15 ml of Protein G (Pharmacia) at a flow rate of 20 ml / min. After further washing of the column with a 150 M NaCl solution, the antibody was eluted with 100 M glycine at pH 3.1. The antibody was isotyped using Mouse Immunoglobulin Immunoglobulin Isotyping Kit (Boehringer Mannheim, Indianapolis, IN) and was found to be a murine IgGl class antibody with a kappa light chain. It has been observed that some isolated proteins are "block" at its terminal amino terminus. By "blocking" it is understood that the amino acid residue of the amino terminal end of the polypeptide sap has been chemically modified in its estrustura after translation, by means of a sealing action or some spontaneous chemical change, in such a way that the polypeptide chain is resistant to the degradation of Edman. Edman's degraded profiling is the chemical procedure routinely used over the last 40 years to determine the amino acid sequence of proteins. The use of the Edman degradation technique, usually automatic in a laboratory instrument conosido somo sequenator or protein sequencer, is a conventional procedure well known to those skilled in the art of protein biochemistry. the conversion of an amino-terminal glutaminyl residue to a pyroglutamyl moiety. This is produced by the cyclization of the glutaminyl moiety to form a structure which is indescerable to the Edman reaction, because a new amide bond is formed between the first alpha-amino group and the delta-carboxyl group. In such cirsunstanaiae, the ability to obtain sesuensia information from the amino-terminal end of the protein depends on the elimination of the pyroglutamyl moiety by a chemical or enzymatic procedure. An important procedure is the use of an enzyme called pyroglutamate aminopeptidase (EC 3.4.19.3) to eliminate the pyroglutamyl moiety. The blocked protein, which may be in solution or electrotransferred to a membrane material such as PVDF (polyvinylidene difluoride) »is treated with a pyroglutamate solution of the protein sufficient to allow the satisfactory determination of the amino acid sequence by chemistry Edman's automatic. Exemplary procedures are described for this in: Fowler EF, Moyer M, Krishna RG, Chin CCQ and Wold F, (1995) "Removal of N-Terminal BlocKing Groups Form Proteins" in Current Protocols in Protein Sciense. , p. 11.7.1-11.7.17 (eds. Coligan JE, Dunn BM, Ploegh HL, Speicher DW. "And Wing ield PT.), John Wiley» New York. In the present work, heavy and light chains of MAb 9A4 were separated by SDS-polysarylamide gel electrophoresis with the use of a reducing agent (beta-mercaptoethanol) in the sample buffer. After electrophoresis, the polypeptides of the gel were electrotransferred to a PVDF membrane and deaged by staining are Coomassie Brilliant Blue R-2SO. The bands containing the heavy and light sahenae of 9A4 were then excised and treated separately with pyroglutamate aminopep idase. In each case »it was shown that it was possible, after this treatment» to obtain information on the amino-terminal sequence »due to Edman degradation. Sequencing was performed on a Procise Model 494 protein sequencer from Perkin-Elmer Applied Biosystems. When it is desired to obtain information on the internal amino acid sequencing (ie, not the N-terminal sesuensia) of the protein, samples transferred from the protein can be digested with trypsin and the resulting digestion product can be fractionated by HPLC to produce individual peptides. that can be sequenced later The following were specific actions of the work: N-terminal unblocking with iroglutamate aminopeptidase (PGAP) The antibody (9A4) was separated into its heavy and light chain constituents by SDS-PAGE on a Tris-Gly polyacrylamide gel 4-20% (Novex, San Diego). It was then transferred over ProBlott (Perkin-Elmer Applied Biosystems, Foster City, CA) and the bands were visualized by tinaion with Ponceau S (Sigma). The membranes containing the light chain of 9A4 were excised and incubated for 30 minutes in a buffer containing 0.1 M sodium phosphate, 10 mM NaaEDTA, 5 mM dithioerythritol. glycerol at 5íí and Triton X-100 reduced to O.IJÍ. Pyroglutamate aminopeptidase (20 mg) (Boehringer Mannheim, Indianapolis, IN) was added to the vial, the contents mixed gently and the reaction was incubated overnight at 37 ° C. The membranes were removed and washed thoroughly in water to remove all salts, detergent and enzyme. Afterwards, the membranes were collated in the protein sessenser Applied Biosystems Model 494 (Perkin-Elmer) to perform the analysis of the N-terminal sequence according to the guidelines provided by the manufacturer. The heavy chain was treated in the same manner as indicated above for the light chain. The following results were obtained from the N-terminal sequence.

Note l: This sequence corresponds to residues 2 to 21 of SEQ ID NO: 6. Note 2. This sequence corresponds to residues 2 to 21 of SEQ ID NO: 5. In addition to the data of the N-terminal sequence previously obtained, the internal sequencing of the light chain of the 9A4 anti- body was also performed, according to the procedure developed by: Fernandez J. Andrews L, Mische SM »Anal Biochem 1994; 218: 112-117. Four bands corresponding to the ProBlott 9A4 light chain were excised and incubated for 30 minutes in a buffer containing 10% acetonitrile and 0.1% Triton X-100 in 0.1 M Tris-HCl pH S.B. Modified Trypsin Grade Modified Trypsin (0.2 mg) (Promega, Madison, Wl) was added and the bands were incubated overnight at 37 ° C. The resulting peptides were extracted from the membrane by washing with acetonitrile at 60 [deg.] C. < »TFA at 0.154, HaO and sonication. The peptides were separated by RP-HPLC on a Vydac C1S 21BTP column (l.O x 250 s) (Vydac »Hesperia» CA). The pyos were visually resuscitated manually and the selessioned peaks were analyzed by automatic Edman sequencing in a Model 494 protein eesuenserver as indicated above. The following results were obtained from the N-terminal sequence for the peptide fragments.

The remains in parentheses can not love as ß tenta iva Note 1: This sequence corresponds to the remains 45 to 60 of the SEQ ID NO: 6. Note 2. This sequel corresponds to residues 61 to 76 of the SEQ ID NO: 6. Note 3. This sequence corresponds to residues 103 to 107 of the SEQ ID NO: 6. Amino Acid Sequence of the Light Chain of 9A4 (V - C) (comparison of the sequence obtained by the sequence of the DNA of the cloned-^ (see below) and the C X.-aj9_e > j murma (obtained by Kabat EA, utt »Perry HM, Bottesman KS and Foeller C-" Sequence of Proteins of Im unologisal Interest, U.S.

Department of Health and Human Services, MIH, 5th Edition, publication N2 91-3242 »1991) with the sequences obtained by the Edman degradation). 1 QIVI.t? SPVg MSASPGBKVT ÜTCSASSSVS? KXWXQQKPG SSPRLIÍI S SUIASCVPVR 61 FSGGGSGTSr SLTISRMZftZ DAATYXCQQVf RSXTRTPGGG trt.v > . t * jt% n ». APTVSIFPPS 121 SEQ TSGGAS WCF NNFYP KDINVKVÍKID GSEROWGVN SWtDQDSKDS TYSMSSTI.TL 181 TKDByERHNS YTCERTHKTS TSPIVKSFNR NEC The underlined amino acids have been sequenced by the automatic sequencing of Edman. The sequence obtained from the cloned DNA, suando is sompara are the amino acid sequences of the fragments of the original antibody protein shown above, indicates that the cloned gene is correct for the V ^ of 9A4. The above amino acid 106 is usually a residue of Lys in the germ line segment Jl, but has been mutated to the sequence shown above for the V ^, while the amino acid 107 (Arg) marks the beginning of the domain C? -, - Cloning and Determination of the Vr, and V "Sequences of MAb 9A4 The hybridoma cell line 9A4 was developed in HT medium (Sigma) are fetal calf serum 554 (Hyslone). Mice were extracted from a cell pellet containing 1? 10"7 cells" using the Pharmacia QuicK Prep mRNA Extraction Kit (Pharmacia) according to the manufacturer's protocol.The cDNA was then synthesized for the gene segments both V ^ and V ,, using the DNA team of Boehringer Mannheim (Indianapolis, IN) according to the protocol provided by the manufacturer.The oligo used in the reaction of the cDNA of, ^ (referred to as MLK) in the List of Discretions as SEQ ID NO: 21 was specific for the Kappa region of the murine light chain "while the oligo used in the reaction of the V cDNA was specific for a segment of the sH3 domain of the gamma region of the murine heavy chain and was designated MHG. in the Sequence List as SEQ ID NO 22. This oligo was designed to bind to four murine IgG isotypes, including IgGl »IgG2a» IgG2b and IgG3 There is a single decoupling of bases for IgGl at nucleotide 5 of SEQ ID NO : 22 pe It will be apparent to those skilled in the art that this would not impede pairing and subsequent generation of the cDNA. Note: The oligos were synthesized by Oligos Etc (Wilsonville, OR), Genosys (The Woodlands »TX) or PerXin Elmer Applied Bioeystems (Foster City, CA>.

The amino terminal amino acid sequence data was used to generate an oligo that could isolate the Bj- gene. of 9A4 correct by PCR, is indicated to sontinuasión. Based on the sequencing of 20 to octosidates presented above for VH, which corresponds to residues 1 to 20 of SEQ ID NO: 5, and assuming that the amino terminal amino acid of the mature form of the heavy chain of the secreted antibody was effectively A residue of Gln 's were compared. Sequences of known antibodies are lae of the VH of 9A4 using the Kafoat database: Sequences of Proteins of Immunological Interest, Volume II, 1991. The domains and H of the antibody corresponding to the first 20 amino acids of 9A4 including: MAb 264 (Nottenburg C »St. John T, and Weissman IL J Immunol 19B7? 139» 1718-1726); MAbRFT2 í Heinrich G, Gram H »Hocher HP, Schreier MH» Ryffel B »AKbar A» A lot PL, and Janossy G, J Immunol 19B9J 143: 3589-3597; and MAb2Hl (Li Y-W, La rie DK, Thammana P, Moore GP and Shearman C W »Mol I munol 1990; 27: 303-311). As it is important to obtain the original DNA sequence corresponding to the eptre or amino terminal of the mature antibody, the oligo PCR 5r must be designed so that it binds 5 '(upstream) of the amino terminal end, that is, in the segment of the signal peptide. The DNA sequences of the three previous antibodies, in which the first 20 amino acids were identical to those of the V--. of 9A4"were sequences with the amino acid sequence and the DNA of the known signal peptide. The DNA sequences are shown below: (The underlined nucleotides indicate differences between the sequences). 264: 5'- GG CTG TGG AAC TTG CTA TTC (SEQ ID MO: 23) RFT2: 5'- GG GTG TGG ACC TTG CCA TTC. { SEQ ID NO: 24) 2H1: 5'- GG GTG TGG ACC TTG CTA TTC (SEQ ID NO: 25) These sequences code for amino acids -11 to -17 of the signal peptides for the, of the so-called antibodies. Therefore, the oligo 5 », MHMISC, was designed to isolate the V? of 9A4 genuine. The sequence of MHMISC is indicated in the Sequence Listing as SEQ ID NO: 26. To isolate the, from 9A4, a series of degenerate oligos was designed to bind to the leader peptide segments of the known murine Kappa light chains. These oligos were used in 5 amplifisasiones by different PCR. (See below). The sesuensies of the degenerated 5 oligoe series were provided by The Dow Chemical Company (Midland MI) and are recognized by the applicant. The oligo 5r that gave a V- ^ of genuine 9A4, was MK3 »whose sequence is indicated in the Sequence List as SEQ ID NO: 27. These oligos encode residues -8 to -14 of the signal peptide. The inverse primers for the ^ and V ,, PCRs were designed from known sequences of the murine IgGl heavy chain constant region (from the C? L domain), called MIGG1CH1 »whose sequence is indicated in the List of Secuensias somo SEQ ID NO: 28, and the constant region of the kappa light chain »denominated MLKN, whose sequencing is indicated in the Sequence List as SEQ ID NO: 8. These oligos were included (sadena above) with respect to the 3T primers used in the synthesis of the cDNA. The reassociation segment begins at nucleotide 10 of SEQ ID NO: 29. The V-H and ^ genes of 9A4 were amplified by PCR using the above-mentioned oligos indicated above and 0.41 μg of the hybridoma cDNA 9A4. PCR was initiated using a GeneAmp1 * Kit with Native Taq Polymerase (Perkin Elmer) and was used according to the manufacturer's specifications. The denaturation, annealing and polymerization temperatures were 94 ° C, 55 ° C and 72 ° C for 45 s, 45 s and 60 s seconds, respectively for the VH PCR. and for the PCR of V ^, »the reassociation temperature was changed to 60 ° C. The PCR was performed for 36 cycles plus a final polymerization cycle of 72 ° C for 7 minutes, followed by a 4 ° C maintenance. The PCR products were cloned into the session room pCR2.1 (Invitrogen, Carlsbad, CA) and sequenced to determine and verify the sequences of ADM and de noacid deduside of the VE, and the Vj ... The oligos used for the determination of the sequence were UNIVLSEQ-5 'and TERMSEQ (-) and are indicated in the Sequence List as SEQ ID NOS: 30 and 31 respectively. The DNA and amino acid sesuensias of V "and ^ of 9A4 are indicated in the Sequence List as SEQ ID NOS: 5 and 6 repectively. The deduced amino acid sequences of the VH and V- ^ 9A4 genes are indicated separately in the List of Sequences as SEQ ID NOS: 32 and 33 respectively. Surprisingly, the oligo MHMISC (SEQ ID NO: 26) satisfactorily provided a V-- sequence, which corresponded exactly to the first 20 amino acids of the mature V H protein determined by the protein sequencing of IgGl 9A4. Immediately 3 'of the oligo PCR 5T »MHMISC (SEQ ID NO: 26), the DNA sequence of the leader peptide segment of the V, of 9A4 is almost identical to that of the corresponding segment of the Mab 2H1» and, therefore, »Probably comes from a V-- gene. they are the same germ line as 2H1. There are only three differences in the amino acid sequence between the 2 antibodies in the V. regions; 1 in CDRl, 1 in CDR2 and 1 in FR3. Probably, these are somatic mutations that have been produced during the affinity maturation process of these two antibodies and can play a role in defining the specificity and affinity of one of the anti- bodies for their respective targets. The V? of 9A4 uses the gene of the murine J "2 binding segment and codes for residues 103 (within CDR3) at 115 of SEQ ID NO: 32. The variable heavy chain of 9A4 belongs to the Family II of heavy chains Ig of kabat mouse. (The V "genes identified here were taken from the Kabat Database at http://immuno.bme.nwu.edu/famgroup.html). The heavy chain V, -. of 9A4 (SEQ ID NO: 5) is very similar to the ID number 001246 of the Database. There are only two diferennsias in these two genes. It appears one in the FRl (silent mutation) and the other in the CDR1 »in the amino acid in position 34 (SEQ ID NO: 32) where the 9A4 has a lie and 001246 has a Met . The most probable is that these two genes come from the same germline of Vj-- .. In this document »H genes of other antibodies related to the V gene are claimed, from the germinal line 9A4» in such a way that when the gene product of VJJ forms a pair - ^ - Vj-. producer of antibodies with the V-L of this other antibody. is one in a way (specificity and affinity) analogous to 9A4. The gene - ^, of 9A4 and the deduced amino acid sequence are indicated in the Sequence list as SEQ ID NOS: 6 and 33, respectively. The deduced amino acid sequence of the fc gene of 9A4 corresponds to all the peptide fragments from the sequencing of the light chain protein of 9A4 genuine somo has been presented above. The variable light chain of 9A4 belongs to the XI Kappa Family of Kabat Mouse and is the most similar to the ID Number 0063O6 of the Database. There are IO disparities of nicleotides that result in 7 amino acid differences. Two of these differences appear in FRl »2 in CDR2, 1 in FR3 and 2 in (CDR3) Co or most of the changes occur in the CDRs, most likely these two genes are related, coming from a V - Germinal line of equal or at least very similar In this document the V, ^ genes of other antibodies derived or related to the V ^ gene of the germinal line 9A4 are claimed, in such a way that when the gene product of V. ^ a V-producing pair of antibodies with the VH of that other antibody, this one is one of a way (specificity and affinity) analogous to 9A 4. Antibodies are also claimed where both V ^ and VH are appropriate. of the genes V ^ and VH of the germinal line 9A4 described in this document, in such a way that when the gene products of ^ and V ^ form a producing pair V - ^ - V- ^ this antibody different from 9A4 is essentially an a way (specificity and affinity) analogous to 9A4 Now we have the base from the c ual design and build versions obtained by genetic engineering of the 9A4 antibody of the present invention. Two examples are indiscriminate, being both antibodies of a single chain (scFv). In one case »the design is V - ^ - linker - V ^ - HIS-MYC marker fragments in the vector pCANTAB6» and in the other the scFv is constructed in the opposite orientation with a different linker »ie VEi-linker- VII-FLAG marker fragment »in the PATDFLAG vector. These examples should not be considered as limiting »but rather as specialists in the G6 technique you will easily realize that domains vt and v? characterized by 9A4 (SEQ ID NOS: 5 and 6) may be used in part or in whole to obtain the previously described types of antibodies, such as Fab »or new ones with configurations using only some critical part of the V. domains.

Construction of 9A4 scFv (Nw-linker-Vr?) In PCANTAB6 PCR primers from SOE (Ayeste por Overlapping Extension) to prepare the assembly of the VH and v * fragments. in a scFv antibody. Two primers were designed and obtained from Perkin- Elmer for the VH region: at the end 5? a site was added Sfi I and at the 3 'end an overlapping sequence was added (3). The VH 5"primer was designated 9A4VH5CAN and the V? 3 'primer was determined to be 9A4H3CAN, which correeponden to SEQ ID NOS: 34 and 35 of the List of Sequences For V ^, a 5T end primer was designed with an overlap in the Glly ^ Ser linker region and a 3 'primer incorporating a Not I restriction site and also obtained from Perkin Elmer. The 5T primer was designated 9A4VL5CAN and the primer of 31 was designated 9A4VL3CANILE, corresponding to SEQ ID NOS: 36 and 37 of the List of Sequences The DNA components of V ,, and V ^ were amplified by PCR and subsequently joined by an assembly step »through SOE-PCR. After the SOE-PCR reaction, an agarose gel band was excised which was identified as a band of -700 pto and gel purified using the QIAquick Gel Purification Kit (QIAgen) in accordance with manufacturer's guidelines. The resulting DNA was directed in Sfi I and Not I and used in a binding with the vector DNA pCANTABd (obtained from Cambridge Antibody Technology, Melburne, Ca bridgeshire, UK), which was also targeted with the same restriction enzymes. Then the ligand DNA was used to transform competent E. coli TG1 cells by electroporation. The basic protocol for generating the competent TG1 E. coli cells was as follows: 500 ml of 2YT medium (biolOl, LaJolla »CA) was preheated at 37 ° C in a 2 1 conical flask and 2 * 5 ml of a recent overnight culture of TG1 cells. These cells were developed with vigorous aeration (300 rpm) at 37 ° C up to OD 600 nm from 0.2 to 0.5, usually from 1 to 1.5 then. The flasks were cooled for 30 minutes on ice, then poured into 250 ml sentrifuge bottles and centrifuged at 4,000 rpm for 15 minutes in a pre-flowed Sorvall centrifuge (4 ° C) to pellet the cells. The cells were resuspended in the original volume of pre-cooled water and centrifuged again as previously unsuspected to pellet the cells. The cells were resuspended in half the original volume, diluted in 250 ml of pre-cooled water and left on ice for 3 minutes, resuspended and centrifuged again as previously indicated. Then »the cells were re-suspended in 20 ml of glycerol at 10 > Pre-cooled, they were transferred to a pre-cooled 50 ml Falcon tube, left on ice for 15 minutes and centrifuged at 3.5O0 rpm, at 4 ° C, for 10 minutes in a Bneshtop centrifuge. Finally the cells were resuspended in 1.0-2.5 ml of glycerol at 10 > 4 pre-cooled and were used directly in the electrolescence stage. The ligated DNA (25-250 ng of ve ctor pCANTAB6-S i I / Not I with VaLV,;, of 9A4-S i I / Not I) was introduced into the E. soli TG1 cells by means of the Stroporasion somo ee indica then: The bound DNA was precipitated with ethanol by the conventional protocol. The DNA probe was dissolved in 10 μl of sterile deionized distilled water. The DNA (up to 1% of the total cell volume) was added to 10Oμl of cells, transferred to a pre-cooled electroporation cuvette (Biorad) and left on ice. The parameters of the Gene Pulser electrophoresis device (Biorad) were set at 25 μFD »2, 5 kV and the impulse controller was set to 200 ohms. The cuvette was dried with a tissue, placed in the electroporation chamber and subjected once to the pulse. Typically, time constants in the range of 3.5-4.8 msec were obtained. The cells subjected to electroporation were immediately diluted with 1 ml of 2YT medium supplemented with 2J4 glucose. The cells were transferred to a 15 ml Falson tube and shaken at 37 ° C for 1 h. The mixture of transformed cells (20-100 μl) was plated on appropriate size plates with agar medium containing 2YT »2% Glucose and 100 μg / ml ampicillin (2YTAG). Initially »we obtained a clone (9A4ICAT3-2) that had a 5 base insertion in the Not I site that placed the downstream sequence out of the phase. To correct this »a new oligo called 9A4NOTFIX3 was made. whose sequence is indicated in the Sequence List as SEQ ID NO: 38. A PCR amplification was analyzed using E. soli cells containing p9A4CAT3-2 as target for the annealing oligos, using the Advantage Klen Taq Polymerase Mix (Clontesh, Palo Alto, CA) according to the manufacturer's protocol. The oligoe of reaeociation (35 moles of each) were 9A4N0TFIX3 and pUCl9R. The sequence of pCU19R is indicated in the Sequence List as SEQ ID NO: 39; chain was annealed upstream of the S i site. The PCR cycles (in a Perkin Elmer Cetus Model 9600 thermal cycloder) were performed as follows: For the first cislo, the DNA denaturation was performed for 1 minute at 94 ° C, annealing for 45 seconds at 60 ° C and polymerization for 1.5 minutes at 68 ° C. For cycles 2-31 »the same temperatures and times were used» with the expectation that the denaturation times were reduced to 30 sec. For the final shekel (32), the polymerization was carried out for 5 minutes "after which" the satellite apparatus cooled the sample to 4 ° C. The TA (Invitrogen) low-tonnage system was used to smelter the resulting produtos. PCR in the plasmid pCR2.1 »using the protosolo suggested by the manufacturer.Twelve white colonies were selected with respect to the inserts using the M13 oligos of Avance and M13 Inverse (invitrogen) by PCR, using the Taq Poli eraea de Klen somo ee has indicated above, three colonies produced inserts of the correct size in agarose gel electrophoresis, one of these was chosen with the correct DNA sequence for the construction V ^ - Engarce-V- ^, of 9A4 for further work. The DNA insert containing the scFv gene was obtained by digestion of the PCR2.1 derivative with Sfi I and Not I, it was purified using the QIAquick Gel Extrastion equipment and the vestige pCANTAB6 digested were the same re-emission enzymes. DNA was introduced into competent E. coli TGl cells by electroporation as described above and resulted in a clone termed P9A4ICAT7-1 (ATCC 98593), which contained an active acFv. The DNA sequence of this asFv of 9A4 is indicated in the Sequence Listing as SEQ ID NO: 7 »while the amino acid sequence is separately indicated as SEQ ID NO: 40. It should be noted that the codon GTG» starting at position 29 of SEQ ID NO: 7 »is the initiation codon (Met). The sequencing oligos used were pUCISR and FDTETSEQ »which are indicated in the Sequence List as SEQ ID NOS: 39 and 41, respectively. See information for SEQ ID MOS: 7 and 40 in the Sequence List, for the specific characteristics of the DNA and amino acid sequences of this scFv antibody. The scFv antibody with V -.-Engarce-v- ^ format of 9A4 obtained by genetic engineering was expressed in E. soli cells.

TG1 and purified using affinity chromatography on NTA-Ni agarose (QIAgen), followed by Seperdex 75 gel permeation chromatography to isolate monomeric scFv species. The binding of the scFv parental antigen was evaluated in the BIAcore (See below). The E. coli containing the plasmid p9A4ICAT7-l has been deposited with the American Type Culture Collection as ATCC-98593.

Constructing ssFv of 9A4 (Vr-engarce-w) in pATDFLAG SOE oligos were also prepared for the assembly by PCR-SOE of fragments VM and V- ^ in the opposite sonfiguration "ie V.¡-engarce-VH, with respect to the example presented previously in pa9a4ICAT7-l. Doe primers were designed for the V - ^ - linker region: at the 5"end a Neo I site was added and at the 3" end an elaspant sesuensia is the 25 amino acid linker, whose sequencing is indicated in the List of Disorders as SEQ ID NO: 42 (Pantoliano MW »Bird RE, Honhnson S. Asel ED, Dodd SW, Wood JF, and Hardman KD Biochemistry 1991; 30: 10117-10125). The primer of Vfc 5rSE DENOMINa 9a4VL5ATD and the primer of L 3T was designated 9A4VL3ATDILE. The sequences of these oligos are indicated in the Sequence List as SEQ ID NOS: 43 and 44, respectively. For VH, a 5 * end primer was designed with an overlap in the linker region and a 3 'end PCR primer to which an Nhe I site had been added. The primer of V, .. 5r was designated 9A4VH5ATD and the VH 3 * primer was designated 9A4VH3ATD. The sequences of these oligos are indicated in the Sequence List as SEQ ID NOS: 45 and 46, respectively. The components V ^ and V? They were amplified by PCR. After they joined V ^ and V? in the linker region by SOE-PCR using the codes 9A4VL5ATD (SEQ ID NO: 44) and 9A4VH3ATD (SEQ ID NO: 46). The DNA was cut to the correct size "of approximately 700 bp" of n 1% agarose gel and eluted using the QIAquick gel eluting kit. The resulting DNA was trimmed at the ends with the restriction enzymes Neo I in the 5 'end and Nhe I at the 3' end. This fragment was bound to the expression vector pATDFLAG (PCT document WO 93/12231) treated are the same restriction enzymes. Competent DHScc E. coli cells were transformed with binding according to the manufacturer's protocol and plated on agar containing 20 μg / ml of chloramphenol as the release agent. Doe of the slones that were sesuenced »P9A4IF-5 and P9A4IF-69 did not have PCR or sonussion errors. The sequencing oligos were: UNIVLSEQ-5? (SEQ ID NO: 30) and TERMSEQ (-) (SEQ ID NO: 31). E. coli cells containing p9A4IF-5 were chosen for further work and for the expression of scFv antibody obtained by genetic engineering. The DNA signaling of this scFv is indicated in the Sequence Listing co or SEQ ID NO: B »while the deduced amino acid sequence is indicated separately in the Sequence Listing as SEQ ID NO: 47. In the Sequence Listing in the information for SEQ ID NOS: 8 and 47 specific characteristics of the V ^ -LV ,, FLAG 9A4 scFv »are provided such as the locations of the signal peptide, linker and marker fragments. It will be evident to those skilled in the art that the antibody obtained by described genetic engineering could be expressed not only in E. coli, but also in other organisms including, but not limited to P. pastoris, Baculovirus, Bacillus species, mammalian cells, etc. . For the expression and purification of this prodrug E. coli ssFv, cultures of 1 »2 liters of cloning LB containing 20 μg / ml of chloramphenicol were allowed to grow overnight at 37 ° C. The cells were pelleted in a Sorvall centrifuge using a GS-3 rotor. In the preparation for affinity chromatography using an M2 affinity column (Kodak, New Haven »CT) (which is specific for the FLAG epitope shown in the previous sequence), E. coli sedi entadae cells were processed in a Tris-EDTA-sasarose medium to isolate the periplasmic fraction or sonicated (Soniprep sonicator) directly in a minimum volume of DPBS buffer. The affinity column was thoroughly washed with DPBS to remove all unbound materials after introducing the crude scFv sample. The scFv antibody was eluted using glycine-HCl 0, 1 M pH 3.1. The monomer was isolated by Superdex-75 gel permeation chromatography (Pharmacia). The antibody was considered homogeneous by SDS-PAGE and staining with Coomassie Brilliant Blue R-250. The antibody was quantified spectrophotometrically at an OD of 2B0 nm »in which it was defined that an absorbance of 1.0 was equivalent to 1.0 mg / ml of scFv» using a superego subste is a trajectory length of 1, 4 cm E. coli containing the pEMEMIDO p9A4IF-5 was deposited with the American Type Culture Collection with ATCC-9B592. The antigen binding in the BIAsore was evaluated for the two purified genie prodrugs p9A4CAT7-l and p9A4IF-5 as indicated below. A streptavidin microplate was introduced with the biotinylated SEQ ID NO: 14 peptide as provided in Example 1 above. The two scFv constructs were shown to bind to the antigen, i.e., in comparison to the parental IgG 9A4 which binds with a K of 1.2 x 10- * 7 M, the 9A4IF-5 has a K of 1, 1 x 10-'7 M. In the case of 9A4ICAT7-1, the inactivation velocity was 1.2 x 10-a sec-3 in comparison are 1.76 x 10-53 sec- for the IgG of 9A4 (parental antibody). These data indicate that the affinity of the antibody obtained by genetic engineering was at least as good or better than the parent. As demonstrated by the speci fi city and kinetic data determined by BIAcore, the two genetically engineered antibodies containing the VL and V domains. of 9A4 described above "has the desired characteristics for use in those in the quantitative measurement assays described in Examples 3 and 4 above. Therefore, other genetically engineered antibodies comprising some or all of the V. ^ and VH domains of 9A4 described herein "and having the affinity and specificity of the parent 9A4 antibody could be considered within the scope of the claims of this invention. .

EXAMPLE 7 Preparation of the engineered antibodies related to 5109 Before cloning the genes of the variable region of 5109 »it was necessary to determine the protein sequence of certain portions of the variable domains of the parent 5109 antibody, so that when the variable domains were cloned, it could be ascertained whether the correct variable domains had been obtained and not others derived from the binding assalate. myeloma or an inactive pseudogene of the B cell used in the generation of the hybridoma. The culture supernatant containing 5109 was generated leaving the hydridome 5109 to cresher in siliceous frassos. The supernatants were adjusted to pH 7.5 with dibasic sodium phosphate and the salt concentration was adjusted with 3 M sodium chloride to a final consentration of 150 mM. The filtered supernatant (O, μ) was passed through a bed volume of 15 ml of Protein G (Pharmacia) at a flow rate of 20 ml / min. After an additional washing of the column with 150 mM NaCl solution. the antibody was eluted with 100 M glycine pH 3.1. The antibody was isotyped using mouse antiserum Iminoglobulin Isotyping Kit (Boehringer Mannheim) and was observed to be an IgG class murine antibody with a constant domain in the kappa light chain. In the present work, the heavy and light chains of the MAb 5109 were separated by SDS-PAGE with the use of a reducing agent (beta-ersaptoethanol) in the sample buffer. After electrophoresis, the polypeptides of the gel were electrotransed to a PVDF membrane and detected by staining with Coomassie Brilliant Blue R-250. The bands containing 5109 heavy and light chains were then excised and subjected to Edman degradation. Sequencing was performed on a Precise Model 494 PerKin-El er Applied Biosystems protein sequencer according to the manufacturer's protocols. The sequences of the heavy and light chains that were obtained are shown in Table 9 and correspond to residues 1 to 40 of SEQ ID NO: 48 for V? and to residues 1 to 39 of SEQ ID NO: 49 for VL.

TABLE 9 Results of the N-terminal amino acid sequence of 5109.

TABLE 9 (CONTINUED) Loe to inoácidoe of attempt are indicated by (). Note: Position 22 of the V, -. and position 23 of the V? it is normally Cys and can not be determined by Edman degradation.

Cloning and determination of the sequences of Vr, and V .. DE MAb 51Q9 Hybridoma cell line 5109 was allowed to grow in HT medium (Sigma) with 5% fetal calf serum (Hyclone). The cells were pelleted (2.5 x 10 7 cells / pellet) and frozen at -80 ° C until use, mRNA extraction was performed (Oligotex ™ Direct mRNA Kit, QIAGEN) according to the guidelines of the Then the cDNA was synthesized for the L and Va regions using the First-Strand cDNA Synthesis Kit from Boehringer Mannheim.The oligo used in the ANDc reaction of V, ^ was specific for the kappa region of the murine light chain (MLK), while the oligo used in the reaction of Vj-c., MGH 'was specific for a segment of the gamma CH- region of the murine heavy chain. The MLK and MHG oligos are indicated in the Sequence List as SEQ ID NOS: 21 and 22. PCR cams were designed for the N-terminal sequencing of the mature secreted forms of the heavy and light chains, based on the sequences of amino acids that were obtained for the ^ and V? by The sequence of the VH 5 'primer and the ^ 5 * primer were designated 51-09V -..-. 5' NDe and Sl-ogv ^ S * NDe respec- tively 'and are indicated in the Sesuena list as SEQ ID NOS 50 and 51. Inverters were designed from known sequences of the heavy chain constant region of IgG1 (for a segment in the CH1 domain) and the constant region of the kappa light chain. The two oligos 3 'eetán in position 5' (upstream) with respect to the original oligos used for the cDNA generator. The V ^ 3r primer, designated MULK2, is indicated in the Sequence List as SEQ ID NOS: 28 and 52, respectively. The resultant products of the PCR for V ^ and V, -. SO joined in pCR2.i and representative clones were chosen for subsequent DNA sequencing. DNA sesuenciation was performed in a Stretsh Model 373 sesuensiador of Applied Biosystems and was carried out in accordance with the protocols provided by the vendor. For the determination of the DNA sequence, the commercial sequencing oligoe M13F and M13R of Invitrogen were used, whose sequences are indissociable in the Sequence List as SEQ ID NOS: 53 and 54. It was observed that the first 21 amino acids were determined by Edman degradation for the mature amino termini of V ^ and VH of 5109 were identical to the corresponding amino acid sequences from the DNA sequence of the corresponding genes that were cloned here by PCR. The sequencing of DNA and deamidaamino acids from the ^ and VH domains of 5109 are indisposed in the List of Sequences as SEQ ID NOS: 10 and 11, respectively. The amino acid sequencies of V ^ and V ^ of 5109 are indicaby • separain the Sequence List as SEQ ID NOS: 48 and 49, respectively. Cinema must be indicawhile the DNA, as indica codes for the correct amino acids of the amino terminal segments of each of the V ^ and V ^ corresponding to the PCR reassociation oligos. The exact codons for the amino acid segments of the antibody corresponding to these oligos "would have appeared in the DNA of the original hybridoma" would not be unambiguous. The variable heavy chain of 5109 is most closely relato the XIV Family of Kabat heavy mouse Ig chains and closely resembles the ID number 002754 of the database. There are 24 nucleotide differences in these 2 VH genes, which result in 14 amino acid differences. Most likely, based on this high number of differences, is that these two genes do not come from a gene of the same germline. However, it is possible that both come from the same germ line and that both have mutain the affinity maturation process in vivo and that the resulting divergence has been amplified. The 5109 gene used for the JK3 binding segment are mutations in the encoder that could otherwise code for a Thr moiety (positions 328 and 330 of SEQ ID NO: 10), but in 5109 it is a ala moiety (position 110). of SEQ ID NO: 48). VH genes are claimed in other antibodies relato the V-* gene of germinal line 5109, such that by adding the prodrug gene from M to a V-2, the prodrug of antibodies is the V ^ of this other anti- body, In 5109, it used a binding segment, which aodifized for the a -ids 102 to 112 as indicain the Sequence List as SEQ ID NOS: 11. The CDR3 for this antibody it is relatively rare because of the Cys-5 residue included in it. CDR3 extends from remainder 94 to 102 of SEQ ID NO: 11 or 49. Cys-94 of SEQ ID NO: 49 was replaced by a Ser residue in a separate gene construct. It was observed that this version had a comparable activity with respect to the scFv that had the parent Cys-94 residue (see below). Val-83 of FR3 of the parental sequence was replaced by Ala ~ 83 of SEQ ID NO: 10 and 49 because of an error in PCR. It was considered unimportant as a conservative difference in terms of the effect on the activity of the products obtained by genetic engineering and, therefore, the ^ of the scFv construction described below was allowed to carry this substitution. The mutation occurred in nucleotide 738 of SEQ ID NO: 9 and resulin a Ala residue in position 215 of SEQ ID NO: 63, of course "for those skilled in the art" it will be apparent that this error of PCR could also be correcand the original Val residue obtained in this position. The V ^ of 5109 belongs to the Kappa VI Family of Kabat Mouse and is very similar to the numbers ID 005841 »005842, 005843 and 005B44 of the database. The 4 antibodies in the database are identical in their nucleotide sequences' so that a comparison of V ^ of 5109 can be made with all four at the same time. There are 12 nucleotide disparities »that result in 8 amino acid differences. One of these differences appears in FRl, 3 in CDR1 »1 in FR2, 1 in CDR2, 1 in FR3 and 1 in CDR3. These changes occur throughout the entire V ^, but focus on the CDRs, with 5 of the 8 differences in these hypervariable segments. Therefore, it is most likely that these genes are related, proceeding from a germ line V ^ equal or at least very similar. Vfc genes are claimed in other antibodies related to the gene - ^ of the germinal line 5109 »in such a way that when the product of the gene V- ^, forms a pair V - ^ - Vj, producer of antibodies with V. -, . of this other antibody, binds in a manner (specificity and affinity) analogous to 5109. Antibodies are also claimed in which both V- ^ and Vs are derived from genes ^ and VH of the germinal line 5109, described in this patent , such that when the gene products of V- ^ and ,, form a V, and V --- producer, this other antibody other than 5109 binds essentially in a manner (specificity and affinity) analogous to 5109.

Construction of the scFv 5109 anti- body obtained by genetic engineering (V ^ -engarce-V-.) In PUC119. PCR savers were used for SOE (Ayuste for Overlapping Extension) to prepare the assembly of the somponents - ^ and VM. All oligos used in the construction of 5109 scFv were synthesized in situ using an Ol? Go 1000 M Beckman DNA synthesizer (Fullerton, CA). These five cabinets, called 5109 VH 5 ', 5109 VH 3', 5109 VL 5 ', 5109 VL 3'SER and 5109 VL 3'CYS are indicated in the Sequence List as SEQ ID NOS: 55 »56» 57 »58 and 59"respectively. The oligo 5109 VL 3 'Ser (SEQ ID NO: 58) was used to change the Cys-94 of SEQ ID NO: 49 to Ser-94 (which corresponds to the Cys residue (SEQ ID NO: 59) to retain the sequence Original Cys After the SOE reaction the DNA was amplified by PCR and the product was sequenced directly to confirm the sequence.The DNA sequence was verified using the Stretsh Model 373 Sequestration Unit from Applied biosystems according to the direstrices of the manufacturer The following oligos were used for the sequencing of potential engineered scFv 5109 antibodies directly linked to pUC119: PUC19R »MycSeqlO» Gly4Ser5 ', Gly4Ser3) f, whose sequences are indicated in the Sequence List as SEQ ID NOS: 39, 60 »61» and 62 »respectively. In the case of the 5109 DNA cassettes bound to the pCR2.1 vector. the oligos M13F and M13R purchased from Invitrogen (SEQ ID NOS: 52 and 53) were used to sequence the priming reactions, together with SEQ ID NOS: 61 and 62 »as two internal oligos. The clones directly linked to pUC119 contained a large number of PCR errors. However, an aseptable clone (H55) was identified in the pCR2.1 vector. This construction was then subaligned by digesting the esFv with Sfi I and Not I and joining it to the vestibule pUC119 digested in a similar way. The slones were ligated with the insertion by PCR amplification using oligos pUC19R and MYsSeq 10 (SEQ ID NOS: 39 and 60).

Three clones underwent sequelae. A slon was identified with the desired sequence and is designated p5109CscFv7 (ATCC); the DNA and deduced amino acid sequence are indicated in the Sequence List as SEQ ID NOS: 9 or 63, respectively. In the Information session for SEQ ID NO: 63, sequence characteristics of this engineered antibody are provided. To verify that the new single-chain constructs retained the binding properties of the parent molecule, the scFv of 5109 was expressed in E. coli and purified. A 2YT starter culture (50 ml) containing 100 μg / ml ampicillin and 2 J4 glucose was inoculated with 50 μl of glycerol stock solution at -BO ° C. The cultures were incubated at 30 ° C with shaking at 330 rpm. Each of six 2 1 flasks containing 2YT medium supplemented with 100 μg / ml ampicillin and 0.1% glochose were inoculated with 5 ml of the starter culture overnight. The cultures were incubated at 30 ° C until they became cloudy and subsequently induced by the addition of IPTG isopropyl B-D-thiogalactopyranoside (Boehringer Mannheim) to a final concentration of 1 mM. The cultures were allowed to grow for 4 more hours for the production of scFv. The cells were centrifuged at 5000 x g for 10 minutes. The cell pellets were stored at -20 ° C until processed. For the purification of scFv, the cell pellets were resuspended in TES (0.2 M Tris-HCl 0.5 mM EDTA »0.5 M sucrose). After resuspension, a 1: 5 dilution of the above TES buffer containing protease inhibitors (Complete Protease Cocktail Inhibitor, Boehringer Mannhei) was added. This preparation was allowed to incubate at 4 ° C for 30 minutes. After incubation, the cells were pelleted at 12,000 x g for 15 minutes. MgCls was added to the resulting supernatant at a final concentration of 5 mM. Ni-NTA agarose (QIAgen) was washed once in PBS containing 300 mM NaCl »15 M imidazole, 0.2% Triton-X, pH 7.4. The washed agarose was then added and the suspension allowed to incubate for 30 minutes at 4 ° C. Then, Ni-NTA + scFv agarose beads were washed 4 times as previously described. Subsequently, the scFv was eluted in washing buffer containing 250 M imidazole. The eluate was desalinated using a NAP-25 (Pharmacia-Biotech »Uppsala» Sweden) solution and concentrated using the Centripep concentrator (Amicon »Berverly» MA). The products were subjected to electrophoresis on SDS-PAGE and visualized by silver staining. The resulting ssFv products for p5109CscFv7 (Cye) and p5109SscFvA9 (Ser) had a purity of approximately 15% and 75%, respectively. Using the Origin methodology (Technical Manual, Origin Instrument »Igen Corporation» Gaithersburg »MD), binding to biotinylated peptide 225 (which has the sequence indicated in the Sequence List as SEQ ID NO: 64) was measured by the scFv species of 5109. Peptide 225 ee was prepared by Anaspec. Peptide 225 was added to 800 μg / ml streptavidin-coated magnetic beads (Dynabeads »Igen» Gaithersburg, MD) to bring the final concentration to 10 nM and incubated for 15 minutes. The new ssFv antibody obtained by genetic engineering was added to the peptide / beads solution for 30 minutes with agitation. Ruttenylated MAb with anti-yc 9E10 marker fragment in 200 μl of Igen assay buffer (Igen, Gaithersburg »MD) was added and the ECL signal was read on the Origin instrument (Igen). MAb 9E10 was generated and purified from the 9E10 line, which was obtained from the ATCC. The MAb was ruteniló using a derivative of N-hidrosuccini ida Origin TAG-NHS Ester (Igen) according to the instructions of the manufacturer. In the arsenic of scFv 5109, a background signal of 2995 ECL units was obtained. The addition of the 5109 scFv Cys construct (p51099CscFv7) resulted in 536,997 ECL units. The addition of construction 5109 scFv Ser (p5109SscFvA9) resulted in 694,253 ECL units. These results demonstrate that the two ssFv of 5109 were biologically astive and bound to the same peptide fragment related to the sologen as a MAb 5109. A sultivo of E. soli £ ue containing p5109CecFvA9 deposited is the American Type Culture Collection co or ATCC-98594 . The specific data determined by the Origin technology demonstrate that the genetically engineered antibody containing the V ^, and V ,, domains of 5109, described above, has the desired characteristics for use in the quantitative measurement assays described above in the examples 3 and 4. Thus, other antibodies obtained by genetic engineering composed of some or all of the domains ^ and V could be considered within the scope of the claims of this invention. of 5109 »described herein, with the affinity and specificity of antibody 5109. It should also be noted that antibodies obtained by genetic engineering are involved in a combination of the V- ^ and V domains. of 9A4 and 5109 »such as a bispecific ssFV dimer of composition: 5109V?: j-Linker-5109VII-Linker-9A4VE-Engar-9A4VI-I ~ Fragment (s) Marker (s)» would also be useful as sole antibody reactive in the previous examples 3 and 4. The difference would be that a single biespesifismo reastivo could be used in a single-stage Eliea instead of uear 9A4 and 5109 separately. For the species in the synthesis it is evident that several genetic compositions comprising the variable domains of the subject antibodies can be attached to obtain a variety of bispecific molecules and thus simplify the assays presented in Examples 3 and 4. The avidity of total molecules can be such that the overall senesity of the assay can also be significantly improved.

EXAMPLE 8 Mutations or differences in the sequences of antibodies to the antibodies relased with the antibodies of the present invention may retain the binding properties (affinity and specificity) and hence the usefulness of the parental antibody. This is demonstrated continuously by generating a series of mutants in the CDR3 of the V--. from. 9A4 ^ ", For those skilled in the art" it is evident that other enzymes in other regions of the V- ^ and VH genes of the same germline "both 9A4 and 5109" can give antibodies are equal or better binding properties than the original anti-bodies show up in this invention.

Generation of utantee in the CDR3 region of? of 9A4 To generate mutants in the CDR3 segment and in 103 Vernier residues immediately adjacent to the CDRα, the pCANTAB6 derivative of ssFv of 9A4 was used, particularly p9A4ICAT7-I presented in Example 6 above. The parental DNA sequence of the CDR3 V ,, region to which the mutation is directed was composed of nucleotides 383 to 409 of SEQ ID NO: 7. The deduced amino acids corresponding to these nucleotides are residues 97 and 105 of the SEQ ID NO: 40. The CDR3 starts at rest 99 and ends at 104. To introduce the random mutations in this area, the V ^, and Vj regions were amplified separately by 2 PCR.

TO In the first PCR. In the first PCR the oligos (obtained from Oligos Etc.) pUC19R (SEQ ID NO: 39) and 9A4MUT (indicated in the Sequence List co or SEQ ID NO: 65) were used to amplify the VH portion "where 9A4MUT was the oligo that introduced the mutations. Nucleotides 25 to 51 of 9A4MUT (SEQ ID NO 65) had a 10% branching. In other words. the written sequence constituted 90% of the nucleotides added to position 25-51"while the other three nucleotides" in each position from 25 to 51, were introduced into the growing oligo chain with a proportion of 3.3% each. This was achieved by well-known procedures in the oligonucleotide synthesis technique. The fact that the random nucleotides were effectively introduced into this defined region will be discussed later. The second PCR used the two oligos (also obtained from Oligos Etc.) 9A4L5 (SEQ ID NO: 66) and FDTETSEQ (SEQ ID NO: 41). This produced the Engarce-V ^ portion on the Not I site of p9A4ICAT7-l at the 3 * end and with an overlap on the V.-, at the 5r end that would allow the subsequent reassociation and assembly of the V ,, products. the PCR mutated coming in the first PCR. PCR was performed as indicated. For the ? mutant, 50 pmoles of each of the oligos pUC19R (SEQ ID NO: 39) and 9A4MUT (SEQ ID NO: 65) were used in reactions of 100 microliters. The template DNA target was an aliquot of P9A4ICAT7-1-plasmid DNA purified by SNAP (Invitrogen). Taq Polymerase (Perkin Elmer) was used in the 30-cycle PCR. The times and temperatures of denaturation, annealing and polymerase reaction were: 94 ° C for 1 minute and 72 ° C for 2 minutes, respectively. For the thirtieth cycle »the polymerization reaction was extended for a total of 10 minutes» before cooling the reaction to 4 ° C. For ^, which would overlap with the VH species, PCR was performed using the Klen Taq Polymerase (Contech) and the Taq Polymerase (Perkin Elmer). The DNA products were purified with the QIAgen gel extraction kit. the assembly reaction by PCR for the v ^ mutated and V ^ was performed using aliquots (1-2 microliters) of each of the purified PCR products in 25 total cycles / 2 temperature cycles: 94 ° C for 1 minute , 65 ° C for 4 minutes »and keeping the temperature at 4 ° C at the end. No oligos were used for this stage. For the PCR extraction of the mutated 9A4 assemblies, 5 μl of a non-purified assembly reaction as template and the oligos pUC19R (SEQ ID NO: 39) and FDTETSEQ (SEQ ID NO: 41) were used for reassortment scavengers. Products of the sorrecto size were observed at approximately 900 bp and gel purified using the QIAgen gel extraction equipment. The mutated 9A4 scFv inserts were treated with Sfi I and Not I to prepare the binding are a DNA of the vector pCANTAB6 cut with the same restriction enzymes. After the union, the DNA mixture was precipitated are ethanol and dissolved in 24 microliters of sterile deionized distilled water. The preparation of the competent E. coli TG1 cells and electrophoration were carried out as described in example 6. 12 different electroporations were made and finally they were combined and plated. A total of 1.5 x 10e clones were obtained and stored as a stock solution in glyceron at -80 ° C. A random sample of 12 clones indicated that 9 of them (one clone did not give the sequence data) had an ineerto, when they were selected by PCR using oligos FDTETSEQ ((SEQ ID NO: 41) and pUC19R (SEQ ID NO: 39 These inserts were purified using the QUIAgen kit and the sequencing of the CDR3 V was determined.The results of the desensitization data are preened to sontinuation.The DNA sequence of the "Parental Sequence" is indicated in the Sequence List. as nucleotides 383 to 409 of SEQ ID NO: 7. No. of Mutations Sequence 5'- GCT AGG GGC GGT AGC CTT GAC TAC TGG -3"Parental: 9A4 UT-1: 5'- GCT CGG GGC GGT AGC CTT GAC TAC CGG -3 '2 9A4MUT-3: 5'- GCT GGG CCC TGT ATC CTT GAJ TAC TGG -3"6 9A4MUT-4: 5'- GCT ACG GGA GGT AGC CTT GAC TAC TGG -3' 2 9A4 UT-6 : 5'- GTT JGG GGC GGC AGC CCT GAC CAC AßG -3 '6 9A4MUT-7: 5'- GCT TGG GGC GGC AGG, JAT GAC TAC TGG -3' 5 9A4MUT-8: 5'- GCT ANG GJC AGT AGC CTT GAC TCC TGG -3 '3 9A4MUT-10: 5'- GCT ACG GGC TCT AGÍ CAT G AC TAC CGC -3 '6 9A4MUT-12: 5'- GCT AGG GGT GGT AGC CTT GAC TAC TGG -3 * 1 Mutations in the previous cohort range from 1 to 6 for each clone and occur in different positions. Mutations are indicated with bold underlined in the various previous clones, N = no nucleotide could be assigned. Only one clone (A4MUT-1) of 8, checked in this random fashion, gave the parental amino acid sequence. Based on the randomness of the previous results, a good mutated library was generated. Therefore, a selection was made with biotin to find the binding to peptide 040 (SEQ ID NO: 14), which is the epitope for 9A4.

Next, description of the biotin selection of the obtained antibodies. An aliquot of approximately 100 μl of stock solution from a mutated library was added to 25 ml of 2YT medium containing 100 μg / ml of ampicillin and 2% glucose. The culture was incubated at 73 ° C for about 60 minutes or until the cells reached the logarithmic middle phase (D 0 βoo = 0.5 to 1.0 = Then the adjuvant phage M13K07 (Pharmacia »Uppsala» Sweden) was added to the culture at a concentration Then the adjuvant phage was allowed to infect the culture for 20 minutes at 37 ° C without agitation and then for a further 25 minutes at 37 ° C with agitation at 200 rpm.The infected cells were transferred. to a 50 ml conical centrifuge tube and sedimented at 3000 rpm for 10 minutes.The cells were resuspended in 2YT medium containing 100 μg / ml ampicillin and 50 μg / ml kanamycin.This culture was transferred to another 250 ml flask. ml and incubated at 30 ° C for 2 hours, during which time phage particles were produced.The cells were removed by centrifugation at 14,000 rpm for 2 minutes, then aliquots (1 ml) of the phage were blocked for 30 minutes. to the t ambient emperature by adding PBS and NFDM to a final concentration of 1 x PBS and 3% NFDM. This was achieved by adding 20O μl of a solution of 6X PBS and 18% NFDM to 1 ml of phage. The biotinylated peptide 040 (SEQ ID NO: 14) was added to the phage solution at concentrations ranging from 10 pM to 1 pM. This solution was incubated for 60 minutes at RT. Magnetic beads coated with streptavidin (Dynal »Oslo» Norway) were blocked at RT with final agitation in 3% NFDM in PBS. After the incubation »the streptavidin beads were captured on the side of the tube with a magnet and the blocking solution was aspirated thoroughly. After the blocked phage was added with the peptide bound to the streptavidin beads and allowed to be incubated at RT, the final agitation was carried out for 15 minutes. The pearl complexes were magnetically captured and the unbound phage was carefully aspirated. The complexes of magnetic linked beads were washed 4 times with PBS containing 0.1% TW-20 and four times with PBS alone. After the final capture, the pear complexes were resuspended in 100 μl of 10 M triethylamine in PBS and neutralized with an equal volume of 1 M Tris pH 7.4. E. coli TG1 cells were then infected in logarithmic phase medium (10 ml) with 100 μl of beads beads. The infection was allowed to progress for another 25 minutes at 37 ° C with shaking at 200 rpm. After the infected cells were pelleted, they were resuspended in 500 μl of fresh 2YT medium and 500 μl was grown on 243 x 243 mm 2YT agar plates containing 100 μg / ml ampicillin and 2% glucose. The plates were incubated overnight at 30 ° C. After approximately 16 hours of growth, the colonies were recovered by scabbing and used to inoculate liquid cultures. The process was repeated and they were inoculated to inoculate liquid cultures. The process was repeated for a minimum of 2 and a maximum of 5 selection rounds. Clones recovered by biotin selection were developed and induced to produce scFv which was purified according to the protocol indicated below.

Preparation of scFv mutant clones using hypnotic shock profusion The culture was pelleted and resuspended in 0.8 ml of ice-cold TES buffer (0.2 M Tris-HCl, 0.5 nM EDTA and 0.5 M sucrose). TES (1.2 ml of 1: 5 dilution cooled with ice) was added and the culture was incubated with ice for 30 minutes.

The cells were pelleted at 4 ° C at 14,000 rpm (30 min.). The supernatant of scFv was added to another tube containing 1 μl of MgCla 1.0 M. 200 μl of NTA-agaroea (QIAgen) was prepared by washing with an imidazole phosphate wash buffer containing 50 mM sodium phosphate »pH 8. O, 500 mM NaCl, 20 mM imidazole and 0.1% TW-20 AL. The supernatant of scFv was added to the NTA-agarose and incubated at 4 ° C for approximately 30 minutes. The NTA-agarose with scFv was centrifuged and 500 volumes of elution buffer were added. The elution buffer consisted of 50 mM sodium phosphate pH 8.0, 500 M MaCl and 250 mM imidazole. The eluate was shaken in a Vortex apparatus and centrifuged to remove the NTA-agarose. The supernatant of ecFv was changed from buffer to a NAP-5 (Pharmacia) column "according to the manufacturer's instructions.

Measurement of inactivation rates for the scFv constructs The inastivation velocities for the sonfussions of esFv were measured by analysis of the dissociation data obtained in the BIAcore (Pharmacia Biosensor) with BIAevaluation software version 2.1. To obtain the data in the BIAcore »streptavidin superfisies were prepared on the BIAcore microspheres as previously described in Example 1. The biotinylated peptide (SEQ ID NO: 14) was attached to the streptavidin surfaces prepared, they were RU densities that varied between approximately 2-11 RU / superfisie. The lower level of derivatization could help to avoid obtaining erroneous inactivation speeds that could be obtained if mass transport were to occur. Purified scFv were injected onto these surfaces to allow binding of the constructs for 60 seconds. The PBS buffer was then replaced alone and the dissociation of scFv was allowed to proceed for 280 sec. plus. Inactivation rates were calculated from this dissociation data.

TABLE 1Q Summary of Vw CDR3 Mutant Sequences of 9 A 4 Amino acid sequences determined from the results of DNA sequencing. The parental sequence for clone ICAT7-1 corresponds to the rest 96 to 105 of DEC ID NO: 40. Speed of "2sec-1" TABLE 10 (continued) Note: Previous ICAT 7-1 and SF-5 clones show CDR3 regions that have the parental sequence. From the data presented above it is evident that sambios can be made in the sequence of amino acids of the parental antiserum still retaining the binding to the target, although the differences in the inactivation rate of the antibodies presented above are within an order of magnitude, addressing to different regions of VH or ^ of the anti- body for the mutation or finding different anti-bodies obtained by unization that have ^ and / or VH domains derived from the V- ^ and V genes? from the same GERMINAL line as 9A4 or 5109, antibodies with variable or improved binding properties can be discovered with respect to the parental antibodies described in Examples 6 and 7 mentioned above.

LIST OF SEQUENCES (1) GENERAL INFORMATION: (iii) NUMBER OF SEQUENCES: 66 (2) INFORMATION FOR SEQ ID NO: 1: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 7 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY; linear (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID 110: 1: Gly Pro Pro Gly Pro Gln Gly 1 5 (2) INFORMATION FOR SEQ ID NO: 2: (i) 'CHARACTERISTICS OF THE SEQUENCE; (A) LENGTH: 7 amino acids (B) TYPE: Amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (ix) CHARACTERISTICS: (A) NAME / KEY: Modified and unusual amino acid (B) LOCATION: 3 (C) OTHER INFORMATION: "Xaa" is 4Hyp (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 2: 1O0 Gly Pro Xaa Gly Pro Gln Gly i 5 (2) INFORMATION FOR SEQ ID NO: 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 10 amino acids (B) TYPE: Amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 3: Gly Glu Pro Gly Asp Asp Gly Pro Ser Gly 1 5 10 (2) INFORMATION FOR SEQ ID NO: 4: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 10 amino acids (B) TYPE: Amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) CHARACTERISTICS: (A) NAME / KEY: Modified and unusual amino acid IB) LOCATION: 3 (C) OTHER INFORMATION: "Xaa" is 4Hyp (xi) SEQUENCE DESCRIPTION: SEQ ID N0: 4: lOl Gly Glu Xaa Gly Asp Asp GLy Pro Ser Gly 1 5 (2) INFORMATION FOR SEQ ID NO: 5: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 408 base pairs (B) TYPE: nucleic acid (C) CHAIN: double (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: adnC (Vi) ORIGINAL SOURCE: (A) ORGANISM: Mus mus caris (ix) FEATURES: (A) NAME / KEY: Signal signal (B) LOCATION: -11 to -1 (C) IDENTIFICATION PROCEDURE : similar to other immunoglobulin signal sequenceshydrophobic (D) OTHER INFORMATION: VH 9A4 region of immunoglobulin (Xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 5. "TTC CTG ATG GCA GCT GCC CAA AGT ATC CAA GCA CAG ATC CAG TTG GTG 48 Phe Leu Mee Ala Ala Ala Gln Be lie Gln Ala Gln lie Gln Leu Val -10 - 5 1 5 CAG TCT GGT CCT GAG CTO AAG AAG CCT GGA GAG GAC ATA GTC AAG ATC TCC 96 Gln Ser Gly Pro Glu Leu Lys Pro Gly Glu Thr Val Lys lie Ser 10 15 20 TGC AAG GCT TCT GGT TAT ACC TTC ACA GAC TAT TCA ATA CAC TGG GTG 144 Cys Lys Wing Ser Gly Tyr Thr Phe Thr Asp Tyr Ser He His Trp Val 25 30 35 AAG CAG GCT CCA GGA AAG GGT TTA AAG TGG ATG GGC TGG ATA AAC ACT 192 Lys Gln Wing Pro Gly Lys Gly Leu Lys Trp Met Gly Trp He Asn Thr 40 45 50 GAG ACT GGT GAG CCA ACTA TAT GCA GAT GAC TTC AAG GGA CGG TTT GCC 240 Glu Thr Gly Glu Pro Thr Tyr Wing Asp Asp Phe Lys Gly Arg Phe Wing 55 60 65 TTC TC GA GAA ACC TCT GCC AGC ACT GCC TAT TTG CAG ATC AAC AAC 288 Phe Ser Leu Glu Thr Ser Wing Be Thr Wing Tyr Leu Gln He Asn Asn 70 75 80 85 CTC AAA AAT GAG GAC ACG GCT ACÁ TAT TTC TGT GCT AGG GGC GGT AGC 336 Leu Lys Asn Glu Asp Thr Wing Thr Tyr Phe Cys Wing Arg Gly Gly Ser 90 95 100 CTT GAC TAC TOG GGC CAA GGC ACC ACT CTC ACA OTC TCC TCA GCC AAA 384 Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Wing Lys 105 110 115 ACG ACÁ CCC CCA TCT GTC TAT CCA 408 Thr Thr Pro Pro Ser val Tyr Pro 120 125 (2) INFORMATION FOR SEQ ID NO: 6: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 359 base pairs (B) TYPE: nusic acid (C) CHAIN: double (D) TOPOLOGY: linear (il) TYPE OF MOLECULE: adnC (vi) ORIGINAL SOURCE: (A) NAME / KEY: signal sequence (B) LOCALIZATION: -7 to -1 (OR IDENTIFICATION PROCEDURE: similar to other immunoglobulin signal sequences » hydrophobic. (D) OTHER INFORMATION: Immunoglobulin V ^ 9A4 region (i) DESCRIPTION OF SEQUENCE: SEQ ID NO: 6: CC TCA GTC ATA CTG TCC AGA GGA 23 Ser Val He Leu Ser Arg Gly -5 CAA ATT GTT CTC ACC CAG TCT CCA GTA TTC ATG TCT GCA TCT CCA GGG 71 Gln He Val Leu Thr Gln Ser Pro Val Phe Met Ser Wing Ser Pro Gly 1 5 10 15 GAG AAG GTC ACC ATG ACC TGC AGT GCC AGC TCA AGT GTA AGT TAC ATG 119 Glu Lys Val Thr Met Thr Cys Ser Wing Ser Ser Ser Val Tyr Met 20 25 30 TAC TGG TAC CAG CAG AAG CCA GGA TCC TCC CCC AGA CTC CTG ATT CAT 167 Tyr Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Arg Leu Leu He His 35 40 45 GCC ACÁ TCC AAC CTG GCT TCT GGA GTC CCT GTT CGC TTC AGT GGC GGT 215 Wing Thr Ser Asn Leu Wing Ser Gly Val Pro Val Arg Phe Ser Gly Gly 50 55 60 GGG TCT GGG ACC TCT TAC TCT CTC ACA ATC AGC CGA ATG GAG GCT GAA 263 Gly Ser Gly Thr Ser Tyr Ser Leu Thr He Ser Arg Met Glu Ala Glu 65 70 75 80 GAT GCT GCC ACT TAT TAC TGT CAG CAG TGG AGA AGT TAT ACA CGG ACG 311 Asp Wing Wing Thr Tyr Tyr Cys Gln Gln Trp Arg Ser Tyr Thr Arg Thr 85 90 95 TTC GGT GGA GGC ACC AAG CTG GAA ATC ATA CGG GCT GAT GCT GCA CCA 359 Phe Gly Gly Gly Thr Lys Leu Glu He He Arg Wing Asp Ala Wing Pro loo 105 110 (2) INFORMATION FOR SEQ ID NO: 7: (A) LENGTH: 883 base pairs (B) TYPE: nucleic acid < C) CHAIN: double (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (ix) CHARACTERISTICS:. { A) NAME / KEY sequence signal (B) LOCATION: -22 to -1 (C) IDENTIFICATION PROCEDURE: similar to other sequences serial »hydrophobic. (i) DESCRIPTION OF SECTION IA: SEQ ID NO: 7: AAGCTTTGGA 10 GCCTTGGAGA TTTTCAAC GTG AAA AAA TTA TTA TC GCA ATT CCT TTA GTT 61 Met Lys Lys Leu Leu Phe Ala He Pro Leu Val - 20 - 15 GTT CCT TTT TAT GCG GCC CAG CCG GCC ATG GCC CAG ATC CAG TTG GTG 109 Val Pro Phe Tyr Wing Wing n Pro Wing Wing Wing Gln lie Gln Leu Val -10 -5 1 5 CAG TCT GGT CCT GAG CTG AAG AAG CCT GGA GAG HERE GTC AAG ATC TCC 157 Gln Ser Gly Pro Glu Leu Lys Pro Gly Glu Thr Val Lys He Be 10 15 20 TGC AAG GCT TCT GGT TAT ACC TTC ACA GAC TAT TCA ATA CAC TGG GTG 205 Cys Lys Wing Ser Gly Tyr Thr Phe Thr Asp Tyr Ser He His Trp Val 25 30 35 AAG CAG GCT CCA GGA AAG GGT TTA AAG TGG ATG GGC TGG ATA AAC ACT 253 Lys Gln Wing Pro Gly Lys Gly Leu Lys Trp Met Gly Trp He Asn Thr 40 45 50 GAG ACT GGT GAG CCA ACTA TAT GCA GAT GAC TTC AAG GGA CGG TTT GCC 301 Glu Thr Gly Glu Pro Thr Tyr Wing Asp Asp Phe Lys Gly Arg Phe Wing 55 60 65 TTC TCT TTG GAA ACC TCT GCC AGC ACT GCC TAT TTG CAG ATC AAC AAC 349 Phe Ser Leu Glu Thr Ser Ala Ser Thr Wing Tyr Leu Gln He Asn Asn 70 75 80 85 CTC AAA AAT GAG GAC ACG GCT ACÁ TAT TTC TGT GCT AGG GGC GGT AGC 397 Leu Lys Asn Glu Asp Thr Wing Thr Tyr Phe Cys Wing Arg Gly Gly Ser 90 95 100 CTT GAC TAC TGG GGC CAA GGC ACC ACT CTC ACA GTC TCC TCA GGT GGA 445 Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Gly Gly 105 110 115 GGC GGC TCA GGC GGA GGC GGC AGC GGC GGC GGC GGA TCG CAA ATT GTT 493 Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Glc GI GI GI GI GI GI GI GI GI GI GI GI GI GI GI GGG GAG AAG GTC 541- Leu Thr Gln Ser Pro Val Phe Met Ser Wing Ser t > ro Gly Glu Lys Val 135 140 145 ACC ATG ACC TGC AGT GCC AGC TCA AGT GTA AGT TAC ATG TAC TGG TAC 589 Thr Met Thr Cys Ser Wing Ser Ser Ser Val Tyr Met Tyr Trp Tyr 150 155 160 165 CAG CAG AAG CCA GGA TCC TCC CCC AGA CTC CTG ATT CAT GCC7 Gln Gln Lys Pro Gly Ser Ser Pro Arg Leu Leu He His Wing Thr Ser 170 175 180 AAC CTG GCT TCT GGA GTC CCT GTT CGC TTC AGT GGC GGT GGG TCT GGG 685 Asn Leu Wing Ser Gly Val Pro Val Arg Phe Ser Gly Gly Gly Ser sly 185 190 195 ACC TCT TCT TCT CTC ACA ATC AGC CGA ATG GAG GCT GAA GAT GCT GCC 733 Thr Ser Tyr Ser Leu Thr He Ser Arg Met slu Ala Glu Asp Ala Ala 200 205 210 ACT TAT TAC TGT CAG CAG TGG AGA AGT TAT ACA CGG ACG TTC GGT GGA 781 Thr Tyr Tyr Cys sln Gln Trp Arg Ser Tyr Thr Arg Thr Phe Gly Gly 215 220 225 GGC ACC AAG CTG GAA ATC ATA GCG GCC GCA CAT CAT CAT CAT CAC CAT CAC 829 Gly Thr Lys Leu Glu He He Ala Ala Ala His His His His His His 230 235 240 245 GGG GCC GCA GAA CAA AAA CTC ATC TCA GAA GAG CTG AAT GGG GCC 877 Gly Ala Ala Glu Gln Lys Leu He Ser Glu Glu Asp Leu Asn Gly Ala 250 255 260 GCA TAG_883_Wing (2) INFORMATION FOR SEQ ID NO: 8: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 1340 paree de baees ÍB) TYPE: nucleic acid (C) CHAIN: double (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (ix) CHARACTERISTICS: (A) NAME / KEY: signal sequence (B) LOCATION: -22 1 -1 (C) IDENTIFICATION PROCEDURE: similar to other signal sequences »hydrophobic.

(Xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: B: CTCATGTTTG ACAGCTTATC ATCGATGAAT TCCATCACTT CCCTCCGTTC ATTTGTCCCC 60 GGTGGAAACG AGGTCATCAT TTCCTTCCGA AAAAACGGTT GCATTTAAAT CTTACATATA 120 TAATACTTTC AAAGACTACA TTTGTAAGAT TTGATGTTTG AGTCGGCTGA AAGATCGTAC 180 GTACCAATTA TTGTTTCGTG ATTGTTCAAG CCATAACACT GTAGGGATAG TGGAAAGAGT 240 GCTTCATCTG GTTACGATCA ATCAAATATT CAAACGGAGG GAGACGATTT TG ATG AAA 298 Met Lys TAC CTA TTG CCT ACG GCA GCC GCT GGA TTG TTA TTA CTC GCT GCC CAA 6 Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala Ala Gln -20 -15 -10 -5 CCA GCC ATG GCC CAA ATT GTT CTC ACC CAG TCT CCA GTA TTC ATG TCT 394 Pro Wing Wing Wing Gln Le Val Leu Thr Gln Ser Pro Val Phe Met Ser 1 5 10 GCA TCT CCA GGG GAG AAG GTC ACC ATG ACC TGC AGT GCC AGC TCA AGT 442 Wing Ser Pro Gly Glu Lys Val Thr Mee Thr Cys Ser Wing Ser Ser 15 20 25 GTA AGT TAC ATG TAC TGG TAC CAG CAG AAG CCA GGA TCC TCC CCC AGA 490 Val Ser Tyr Met Tyr Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Arg 30 35 40 CTC ATTO CAT GCC ACÁ TCC AAC CTG GCT TCT GGA GTC CCT GTT CGC 538 Leu Leu He His Wing Thr Ser Asn Leu Wing Ser Gly Val Pro Val Arg 45 SO 55 60 TTC AGT GGC GOT GGG TCT GGG ACC TCT TAC TCT CTC ACA ATC AGC CGA 586 Phe Ser Gly Gly Gly Ser Gly Thr Ser Tyr Ser Leu Thr He Be Arg 65 70 75 ATG GAG GCT GAA GAT GCT GCC ACT TAT TAC TGT CAG CAG TGG AGA AGT 634 Mee Glu Wing Glu Asp Wing Wing Thr Tyr Tyr Cys Gln Gln Trp Arg Ser 80 85 90 TAT ACÁ CGG ACG TTC GGT GGA GGC ACC AAG CTG GAA ATC ATA CTT AGT 682 Tyr Thr Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu lie He Leu Ser 95 100 105 GCG GAC GAT GCG AAA AAG GAT GCT GCG AAG AAG GAT GAC GCT AAG AAA 730 Wing Asp Asp Wing Lys Lys Asp Wing Wing Lys Lys Asp Asp Wing Lys Lys 110 115 120 GAC GAT GCT AAA AAG GAC CTC GAG ATC CAG TTG GTG CAG TCT GGT CCT 778 Asp Asp Ala Lys Lys Asp Leu Glu He Gln Leu Val Gln Ser Gly Pro 125 130 135 140 GAG CTG AAG AAG CCT GGA GAG HERE GTC AAG ATC TCC TGC AAG GCT TCT 826 Glu Leu Lys Lys Pro Gly Glu Thr Val Lys He Ser Cys Lys Wing Ser 145 150 155 GGT TAT ACC TTC ACÁ GAC TAT TCA ATA CAC TGG GTG AAG CAG GCT CCA 874 Gly Tyr Thr Phe Thr Asp Tyr Ser He His Trp Val Lys Gln Wing Pro 160 165 170 GGA AAG GGT TTA AAG TGG ATG GGC TGG ATA AAC ACT GAG ACT GGT GAG 922 Gly Lys Gly Leu Lys Trp Mee Gly Trp He Asn Thr Glu Thr Gly Glu 175 180 185 CCA ACTA TAT GCA GAT GAC TTC AAG GGA CGG T T GCC TTC TCT TTG GAA 970 Pro Thr Tyr Wing Asp Asp Phe Lys Gly Arg Phe Wing Phe Ser Leu Glu 190, 195 200 ACC TCT GCC AGC ACT GCC TAT TTG CAG ATC AAC AAC CTC AAA AAT GAG 1018 Thr Ser Ala Ser Thr Wing Tyr Leu Glp He Asn Asn Leu Lys Asn Glu 205 210 215 220 GAC ACG GCT ACÁ TAT TTC TGT GCT AGG GGC GGT AGC CTT GAC TAC TGG 1066 Asp Thr Wing Thr Tyr Phe Cys Wing Arg Gly Gly Ser Leu Asp Tyr Trp 225 230 235 GGC CAA GGC ACC ACT CTC ACÁ GTC TCC TCA GCT AGC GAC TAC AAG GAC 1114 Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Wing Ser Asp Tyr Lys Asp 240 245 250 GAT GAT GAC AAA TAAAAACCTA GCGATGAATC CGTCAAAACA TCATCTTACA 1166 Asp Asp Asp Lys 255 TAAAGTCACT TGGTGATCAA GCTCATATCA TTGTCCGGCA ATGGTGTGGG CTTTTTTTGT 1226 TTTCTATCTT TAAAGATCAT GTGAAGAAAA ACGGGAAAAT CGGTCTGCGG GAAAGGACCG 1286 GGTTTTTGTC GAAATCATAG GCGAATGGGT TGGATTGTGA CAAAATTCGG ATCC 1340 IOS (2) INFORMATION FOR SEQ ID NO: 9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 907 base pairs (B) TYPE: CC nucleic acid) CHAIN: double (D) TOPOLOGY: linear Cii) TYPE OF MOLECULE: DNA (IX) CHARACTERISTIC: (A) NAME / KEY: signal sequence (B) LOCATION: -22 to -1 (C) IDENTIFICATION PROCEDURE: similar to other signal sequences »hydrophobic. (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 9: AAGCTTTGGA 10 GCCTTGGAGA TTTTCAAC GTG AAA AAA TTA TTA TTC GCA ATT CCT TTA GTT 61 Met Lys Lys Leu Leu Phe Wing He Pro Leu Val -20 -15 GTT CCT TTT TAT GCG GCC CAG CCG GCC ATG GCC GAA GTG CAG CTG GTG 109 Val Pro Phe Tyr Wing Wing Gln Pro Wing Wing Wing Glu Val Gln Leu Val -10 -5 1 5 GAG TCT GGG GGA GGC TCA GTG CAG CCT GGA GGG TCC CTG AAA CTC TCC 157 Glu Ser Gly Gly Gly Ser Val Gln Pro Gly Gly Ser Leu Lys Leu Ser 10 15 20 TGT GCA GCC TCT GGA TTC ACT TTC AAT ACC TAC OGC ATG TCT TOG GTT 205 Cys Wing Wing Ser Gly Phe Thr Phe Asn Thr Tyr Gly Met Ser Trp Val 25 30 35 CGC CAG ACT CCA GAC AAG AGG CTG GAG TGG GTC GCA ACC ATT AAT AGT 253 Arg Gln Thr Pro Asp Lys Arg Leu Glu Trp Val Wing Thr He Asn Ser 40 45 50 AAT GGT GGT CTC ACC TTT TAT GCA GAC AGT GTG AAG GGC COA TTC ACC 301 Asn Gly Gly Leu Thr Phe Tyr Wing Asp Ser Val Lys Gly Arg Phe Thr 55 60 65 ATT TCC AGA GAC AAT GCC AAA AAC ACC CTG TAT CTG CAA ATG AAC AGG 349 He Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu G n Met Asn Arg 70 75 80 85 CTG AAG TCT GGG GAC TCA GGC ATG TAT TAC TGT GTA AGA GGA TAT AGT 397 Leu Lys Ser Gly Asp Ser Gly Met Tyr Tyr Cys Val Arg Gly Tyr Ser 90 95 100 AAT TAC GCT CGC TGG GGC CAA GGG GCG CTG GTC ACT GTC TCG AGT GGT 445 Asn Tyr Wing Arg Trp Gly Gln Gly Wing Leu Val Thr Val Ser Ser Gly 105 110 115 GGA GGC GGT TGA GGC GGA GGT GGC AGC GGC GGT GGC GGA TCG TCT GAT 493 Gly Gly Gly Ser Gly Gly Gly Gly Gly Gly Gly Gly Ser Being Asp 120 12S 130 GTT GTG ATG ACC CAA ACT CCA ACT TTG TCG GTT ACC ATT GGA CAA 541 Val Val Met Thr Gln Thr Pro Leu Thr Leu Ser Val Thr He Gly Gln 1 35 140 145 TCA GCC TCC ATC TCT TGC AAG TCA AGT CAG AGC CTC TTA GGT AGT GAT 589 Ser Wing Ser He Ser Cys Lys Ser Ser Gln Ser Leu Leu Gly As Asp 150 155 160 165 GGA TTG ACÁ TAT TTG ATT TGG TTG TTG CAG AGG CCA GGC CAG TCT CCA 637 Gly Leu Thr Tyr Leu He Trp Leu Leu sln Arg Pro Gly Gln Ser Pro 170 175 180 AAG CGC CTA ATC TTT CTG GTG TCT GAA TTG GAC TCT GGA GTC CCT GAC 685 Lys Arg Leu He Phe Leu Val Ser Glu Leu Asp Ser Gly Val Pro Asp 185 190 195 AGG TTC ACT GGC AGT GGA TCA GGG ACÁ GAT TTC AA CTG AAA ATC AGC 733 Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys He Ser 200 205 210 AGA GCG GAG GCT GAA GAT TTG GGA GTT TAT TAT TGC TGC CAG GGT ACÁ 781 Arg Wing Glu Wing Glu Asp Leu Gly Val Tyr Tyr Cys Cys Gln Gly Thr 215 220 225 CAT TTT CCT CAC ACG TTC GGT GCT GGG ACC AAG CTO GAG CTG AAA GCG 829 His Phe Pro His Thr Phe Gly Wing Gly Thr Lys Leu Glu Leu Lys Wing 230 235 240 245 GCC GCA GAA CTA AAA CTC ATC TCA GAA GAG GAT CTO AAT GGG GCC GCA 877 Wing Ala Glu Gln Lys Leu He Ser Glu Glu Aep Leu Asn Ala Ala Ala 250 255 260 CAT CAC CAC CAT CAC CAT TAATAAGAAT TC 907 His His His His His His 265 His. (2) INFORMATION FOR THE? EC ID NO: 10: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 34B base pairs (B) TYPE: nucleic acid CC) CHAIN: double (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: adnC (ix) CHARACTERISTICS: (A) NAME / KEY: VH monoclonal antibody 5109 (B) LOCATION: 1 to 116 (Ki) DESCRIPTION OF SEQUENCE: SEQ ID NO: 10: GAA GTG CAG CTG GTG GAG TCT GGG GGA GGC TCA GTG CAG CCT GGA GGG 48 Glu Val Gln Leu Val slu Ser Gly Gly Gly Ser Val Glp Pro Gly Gly 1 5 10 15 TCC CTG AAA CTC TCC TGT GCA GCC TCT GGA TTC ACT TTC AAT ACC TAC 96 Ser Leu Lys Leu Ser Cys Wing Wing Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 GGC ATG TCT TGG GTT CGC CAG ACT CCA GAC AAG AGG CTG GAG TGG GTC 114 Gly Mee Ser Trp Val Arg Gln Thr Pro Asp Lys Arg Leu Glu Trp Val 35 40 45 GCA ACC ATT AAT AGT AAT GGT CTC ACC TTT TAT GAC GAC AGT GTG 192 Wing Thr He Asn Ser Asn Gly Gly Leu Thr Phe Tyr Wing Asp Ser Val 50 55 60 AAG GGC CGA TTC ACC ATT TCC AGA GAC AAT GCC AAA AAC ACC CTG TAT 240 Lys Gly Arg Phe Thr He Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 CTG CAG ATG AAC AGG CTG AAG TCT GGG GAC TCA GGC ATG TAT TAC TGT 288 Leu Glp Met Asn Arg Leu Lye Ser Gly Asp Ser Gly Met Tyr Tyr Cys 85 90 95 1 GTA AGA GGA TAT AGT AAT TAC GCT CGC TGG GGC CAA GGG GCG CTG GTC 336 Val Arg Gly Tyr Ser Asn Tyr Ala Arg Trp Gly sln Gly Ala Leu Val 100 105 110 ACT GTC TCT GCA 348 Thr Val Ser Wing 115 (2) INFORMATION FOR SEQ ID NO: 11: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 336 paree of bases (B) TYPE: nucleic acid (C) CHAIN: double (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: adnC (ix) CHARACTERISTICS: (A) NAME / KEY: V- ^ of monoclonal antibody 5109 (B) LOCATION: 1 to 112 (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 11: GAT GTT GTG ATG ACC CAA ACT CCA ACT TTG TCG GTT ACC ATT GGA 48 Asp Val Val Met Thr Gln Thr Pro Leu Thr Leu Ser Val Thr He Gly 1 5 10 15 CAA TCA GCC TCC ATC TCT TGC AAG TCA AGT CAG AGC CTC TTA GGT AGT 96 Gln Ser Wing Be Ser Cys Lys Ser Ser Gln Ser Leu Leu Gly Ser 20 25 30 GAT GGA TTG ACÁ TAT TTG ATT TGG TTG CAG AGG CCA GGC CAG TCT 144 A3p Gly Leu Thr Tyr Leu He Trp Leu Leu Gln Arg Pro Gly Gln Ser 35 40 45 CCA AAG CGC ATC TTT CTG GTO TCT GAA TTG GAC TCT GGA GTC CCT 192 Pro Lys Arg Leu He Phe Leu Val Ser Glu Leu Asp Ser Gly Val Pro 50 55 60 GAC AGG TTC ACT GGC AGT GGA TCA GGG AC GAT TTC ACA CTG AAA ATC 240, Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys He 65 70 75 80 AGC AGG GTG GAG GAT GAA GAT TTG GGA GTT TAT TAT TGC TGC CAG GGT 288 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Cys Gln Gly 85 90 95 ACÁ CAT TTT CCT CAC ACG TTC GGT GCT GGG ACC AAG CTG GAG CTG AAA 336 Thr His Phe Pro His Thr Phe Gly Wing Gly Thr Lys Leu Glu Leu Lys 100 105 110 n; (2) INFORMATION FOR SEQ ID NO: 12: (I) SEQUENCE CHARACTERISTICS: (A) LENGTH: 13 amino acids (B) TYPE: amino acids (O TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (ix) CHARACTERISTICS: (A) NAME / KEY: modified and unusual amino acid (B) LOCATION: 2 (C) OTHER INFORMATION: "Xas" is 4Hyp (i) CHARACTERISTICS: (A) NAME / KEY: modified and unusual amino acid (B) LOCA IZATION: 8 (C) OTHER INFORMATION: "Xaa" ee 4Hyp (i) CHARACTERISTICS: (A) NAME / KEY: modified and unusual amino acid (B) LOCATION: 11 (C) OTHER INFORMATION: "Xaa'1 ee 4Hyp (xi) ) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 12: Ala Xaa Gly Glu Asp Gly Arg Xaa Gly Pro Xaa Gly Pro x 5 10 (2) INFORMATION FOR SEO ID NO: 13: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 20 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (ix) CHARACTERISTICS : (A) NAME / KEY: modified and unusual amino acid (B) LOCATION: 9 (C) OTHER INFORMATION: "Xaa." ee 4Hup (i) CHARACTERISTICS: (A) NAME / KEY: modified and unusual amino acid (B) LOCATION: 15 (C) OTHER INFORMATION: "Xaa" ee 4Hyp (ix) CHARACTERISTICS: (A) NAME / KEY: modified amino acid e unusual (B) LOCATION: IB (C) OTHER INFORMATION: "Xaa" ee 4Hyp (ix) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 13: Gly Lys Val Gly Pro Ser Gly Ala Xaa Gly s'lu Asp Gly Arg Xaa Gly 1 5 10 15 Pro Xaa Gly Pro 20 (2) INFORMATION FOR SEQ ID NO: 14: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH : 9 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (Xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 14: Wing Glu Gly Pro Pro Gly Pro Gln Gly 1 5 (2) INFORMATION FOR SEQ ID NO: 15: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 10 aminoacid (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (i) CARACTER ISTICS: (A) NAME / KEY: modified and unusual amino acid (B) LOCATION: 3 (OR OTHER INFORMATION: "Xaa" is 4Hyp (i) CHARACTERISTICS: (A) NAME / KEY: modi ised and unusual amino acid (B) LOCALIZATION : 7 (C) OTHER INFORMATION: solagenaea essindida in the COOH site of residue 7 (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 15: Gly Pro xaa Gly Pro sln Gly Leu Ala Gly 1 5 10 (2) INFORMATION FOR SEQ ID NO: 16: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 7 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (Xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 16: Gly Thr Pro Gly Pro Gln Gly (2) INFORMATION FOR SEQ ID NO: 17: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 10 aminoasidoe (B) TYPE: aminoaside (D) linear TOPOLOGY (ii) TYPE OF MOLECULE: peptide (Xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 17: Cys Ala Glu Gly Pro Pro Gly Pro Gln Gly 1 5 10 (2) INFORMATION FOR SEQ ID NO: 18: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: IO to inoacid (B) TYPE: amino acid (D) TOPOLOGY : linear (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 18: Cys Gly Glu Pro Gly Asp Asp Gly Pro Ser c 10 (2) INFORMATION FOR SEQ ID NO: 19: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 9 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 19: Gly Glu Pro Gly Asp Asp Gly Pro Ser (2) INFORMATION FOR SEQ ID NO: 20: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 19 amino acids (B) TYPE: amino acid (D> TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 20: Gly Glu Pro Gly Asp Asp Gly Pro Be Gly Wing Glu Gly Pro P or Gly 1 5 10 -5 Pro Gln Gly (2) INFORMATION FOR SEQ ID NO: 21: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 20 amino acids (B) TYPE: nucleic acid (OR simple CHAIN (D) TOPOLOGY: linear (i) DESCRIPTION OF THE SEQUENCE : SEQ ID NO: 21: GGTGAAGTTG ATGTCTTGTG 20 (2) INFORMATION FOR SEQ ID NO: 22: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 23 amino acids (B) TYPE: nucleic acid (OR simple CHAIN (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE : SEQ ID NO: 22: GACCTTGCAT TTGAACTCCT TGC 23 (2) INFORMATION FOR SEQ ID NO: 23: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (OR simple CHAIN (D) TOPOLOGY: linear ( xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 23: GGCTGTGsAA CTTGCTATTC 20 (2) INFORMATION FOR SEQ ID NO: 24: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 20 base pairs ÍB) TYPE: nusleiso acid (C) sensella CHAIN (D) TOPOLOGY: linear (i). DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 24: GGGTGTGGAC CTTOCCATTC _ 20 (2) INFORMATION FOR SEQ ID NO: 25: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) single CHAIN (D) TOPOLOGY: 1inea1 (Xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 25: GGGTGTGGAC CTTGCTATTC (2) INFORMATION FOR SEQ ID NO: 26: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) single CHAIN ID) TOPOLOGY: linear (i) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 26: GGSTGTGGAM CTTGCYATTC (2) INFORMATION FOR SEQ ID NO: 27: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: IB base pairs (B) TYPE: nusleiso rough (C) sensilla CHAIN (D) TOPOLOGY: linear (i) DESCRIPTION OF THE SEQUENCE: SEQ ID NQ: "27: 18 GCTTCCTOCT AATCAKTG (2) INFORMATION FOR SEQ ID NO: 28: (i) SEQUENCE CHARACTERISTICS (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (OR simple CHAIN (D) TOPOLOGY: linear (i) DESCRIPTION OF THE SEQUENCE: SEO ID NO: 28: 21 GGCAGCAGAT CCAGGGGCCA G (2) INFORMATION FOR SEQ ID NO: 28: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) Simple CHAIN (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 29: 33 GGGAAAGCTT GTTAACTGCT CACTGGATGG TGG (2) INFORMATION FOR SEQ ID NO: 30: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (OR simple CHAIN (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 30: 22 CTATTGCCTA CGGCAGCCGC TG (2) INFORMATION FOR SEQ ID NO: 31: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) single CHAIN (D) linear TOPOLOGY (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 31: CACATGATCT TTAAAGATAG (2) INFORMATION FOR SEQ ID NO: 32: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 136 base pairs (B) TYPE: nucleic acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE porteine (i) ) CHARACTERISTICS: (A) NAME / KEY: portion of the VH signal peptide of 9A4_ (B) LOCATION: -11 to -1 (OR IDENTIFICATION PROCEDURE: comparison with another and murine immunoglobulins (i) FEATURES: (A) NAME / KEY : mature protein a of SA4 (B) LOCATION: 1 to 115 (C) IDENTIFICATION PROCEDURE: amino-terminal sequencing »comparison with other pturine immunoglobulins (i) CHARACTERISTICS: (A) NAME / KEY: beginning of the CH-L domain of murine IgGl (B) LOCATION: 116 to 125 (OR IDENTIFICATION PROCEDURE: somparation with other murine IgG immunoglobulins (i) DESCRIPTION OF SEQUENCE: SEQ ID NO: 32: phe Leu Met Ala Ala Ala Ala Gln Ser lie Gln Ala Gln He Gln Leu Val -10 -5 1 s Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu Thr Val Lys He Ser 10 15 20 Cys Lys Wing Ser Oly Tyr Thr Phe Thr Asp Tyr Ser lie His Trp Val 25 30 35 Lys Gln Wing Pro Gly Lys Gly Leu Lys Trp Met Gly Trp lie Asn Thr 40 45 50 Glu Thr Gly Glu Pro Thr Tyr Wing Asp Asp Phe Lys Gly Arg Phe Wing 55 SO 65 Phe Ser Leu Glu Thr Ser Ala Ser Thr Wing Tyr Leu Gln lie Asn Asn 70 75 80 85 Leu Lys Asn Glu Asp Thr Wing Thr Tyr Phe Cys Wing Arg Gly Gly Ser 90 95 100 Leu Asp Tyr Trp Gly Gln Thr Thr Thr Leu Thr Val Ser Ser Wing Lys 105 110 115 Thr Thr Pro Pro Ser Val Tyr Pro (2) INFORMATION FOR SEQ ID NO: 33: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 119 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (ix) CAR CTE IS ICAS: (A) NAME / KEY: portion of the V- ^ signal peptide of 9A4 (B) LOCATION: -1 to (C) IDENTIFICATION PROCEDURE: somparation with other murine immunoglobulins (ix) FEATURES: (A) NAME / KEY: protein mature V ^ of 9A4 (B) LOCATION: 1 to 106 (C) IDENTIFICATION PROCEDURE: terminal amino sequences »comparison with other rxturin immunoglobulins (xi) DESCRIPTION OF SEQUENCE: SEO ID NO: 33: Ser Val lie Leu Ser Arg Glv Gln lie Val Leu Thr Gln Ser Pro Val -5 1 s Phe Met Ser Wing Pro Gly Glu Lys Val Thr Met Thr Cys Ser Wing 10 15 20 25 Ser Ser Val Val Tyr Met Tyr Trp Tyr Gln Gln Lys Pro Gly Ser 30 35 40 Pro Pro Arg Leu Leu lie His Wing Thr Ser Asn Leu Wing Ser Gly Val 45 50 55 Pro Val Arg Phe Ser Gly Gly Gly Ser Gly Thr Ser Tyr Ser Leu Thr 60 65 70 lie Ser Arg Met Glu Wing Glu Asp Wing Wing Thr Tyr Tyr Cys Gln Gln 75 ao 85 Trp Arg Ser Tyr Thr Arg Thr Phe Gly Gly Gly Thr Lys Leu slu lie 9 ° '95 100 ios lie Arg Ala Asp Ala Ala Pro 110 (2) INFORMATION FOR SEQ ID NO: 34: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 47 base pairs (B) TYPE: nucleic acid (C) Simple CHAIN (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 34: GAGGAGGCCC AGCCGGCCAT GGCCCAGATC CAGTTGGTGC AGTCTGG 47 (2) INFORMATION FOR SEQ ID NO: 35: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 61 base pairs (B) TYPE: nucleic acid (C) single CHAIN (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 35: GCCGCTGCCA CCTCCGCCTG AACCGCCTCC ACCACTCGAG ACTGTGAGAG TGGTGCCTTG 60 61 (2) INFORMATION FOR SEQ ID NO: 36: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 56 base pairs (B) TYPE: nucleic acid (C) Simple CHAIN (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 36: GGTTCAOsCG sAGGTGGCAs CssCGGTOGC GGATCGCAAA TTGTTCTCAC CCAGTC 56 (2) INFORMATION FOR SEQ ID NO: 37: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 3B pairs of toases (B) TYPE: nucleic acid ÍC) Simple CHAIN (D) TOPOLOGY: linear (Xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 37: GAAGGACGCC GGCGTATGAT TTCCAGCTTG GTGCCTCC 38 (2) INFORMATION FOR SEQ ID NO: 38: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 36 base pairs (B) TYPE: nucleic acid (C) Simple CHAIN (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 38; -_Q AAGAAGCGGC CGCTATGATT TCCAGCTTGG TGCCTC 36 (2) INFORMATION FOR SEQ ID NO: 39: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 23 base pairs 15 (B) TYPE: nucleic acid ÍC) Simple CHAIN (D) TOPOLOGY: linear (Xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 39: 0 AGCGGATAAC AATTTCACAC AGG 23 (2) INFORMATION FOR SEQ ID NO: 40: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 2B4 amino acids 5 (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (ix) CHARACTERISTICS: ÍA) NAME / KEY: signal peptide pCANTAB6 (B) LOCATION: -22 A -1 ÍC) IDENTIFICATION PROCEDURE: hydrophobic (ix) CHARACTERISTICS: (A) NAME / KEY: Mature protein scFv of 9A4 »Vj -.- engarce- ^ -marker segments (B) LOCATION: 1 to 262 (i) CHARACTER IS ICAS: (A) NAME / KEY: V? of 9A4 < B) LOCATION: 1 to 115 (i) CHARACTERISTICS: (A) NAME / KEY: link (Gly ^ Ser) 3 (B) LOCATION: 116 to 130 (ix) FEATURES: (A) NAME / KEY: V ^ of 9A4 (B) LOCATION: 131 to 236 (i) CHARACTERISTICS: (A) NAME / KEY: His segment (B) LOCATION: 240 to 245 (ix) FEATURES: (A) NAME / KEY: myc segment (B) LOCATION: 249 a 25B (X i) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 40 Mee Lys Lys Leu Leu F .-. E Ala l ie Pro Leu Val Val Pro Phe Tyr Ala - 20 - 15 - 10 Ala Gln Pro Ala Met Ala G n l ie Gln Leu Val Gln Ser Gly Pro Glu -5 1 5 10 Leu Lys Lys Pro Gly Gl-i Thr Val Lys lie Ser Cys Lys Wing Ser Gly 15 20 25 Tyr Thr Phe Thr Asp Tyr Ser lie His Trp Val Lys Gln Wing Pro Oly 30 35 40 Lys Gly Leu Lys Trp Mee Gly Trp He Asn Thr Glu Thr Gly Glu Pro 45 50 55 Thr Tyr Wing Asp Asp Phe Lys Gly Arg Phe Wing Phe Ser Leu Glu Thr 60 65 70 Wing Wing Thr Wing Tyr Leu Gln He Asn Asn Leu Lys Asn slu Asp 75 63 85 90 Thr Ala Thr Tyr Phe C / s Wing Arg Gly Gly Ser Leu Asp Tyr Trp Gly 95 100 105 Gln Gly Thr Thr Leu Thr Val Ser Gly Gly Gly Gly Ser Gly Gly 110 115 120 Gly Gly Ser Gly Gly Gly Ser Gln He Val Leu Thr Gln Ser Pro 125 130 135 Val Phe Met Ser Wing Ser Pro Gly Glu Lys Val Thr Met Thr Cys Ser 140 145 150 Wing Being Ser Val Val Tyr Met Tyr Trp Tyr Gln Gln Lys Pro Gly 155 160 165 170 Ser Ser Pro Arg Leu Leu He His Wing Thr Ser Asn Leu Wing Ser Gly 175 180 185 Val Pro Val Arg Phe Ser Gly Gly Gly Ser Gly Thr Ser Tyr Ser Leu 190 195 200 Thr He Ser Arg Met Glu Wing Glu Asp Wing Wing Thr Tyr Tyr Cys Gln 205 210 215 Gln Trp Arg Ser Tyr Thr Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu 220 225 230 He He Ala Ala Ala His His His His His His Gly Ala Ala Glu Gln 235 240 245 250 Lys Leu He Ser Glu Glu Asp Leu Asn Gly Ala Ala 255 260 (2) INFORMATION FOR SEQ ID NO: 41: (i) CHARACTERISTICS OF THE SEQUENCE ÍA) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) single CHAIN ÍD) TOPOLOGY: linear (Xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 41: GTCGTCTTTC CAGACGTTAG T 21 (2) INFORMATION FOR SEQ ID NO: 42: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 25 base pairs (B) TYPE: nucleic acid ÍC) Simple CHAIN (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 42: Leu Ser Wing Asp Asp Wing Lys Lys Asp Wing Wing Lys Lys Asp Asp Wing 5 10 15 Lys Lys Asp Asp Wing Lys Lys Asp Leu 20 25 (2) INFORMATION FOR SEQ ID NO: 43: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 34 base pairs ÍB) TYPE: nucleic acid (C) single CHAIN (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 43: 34 GAGGAGCCAT GGCCCAAATT GTTCTCACCC AGTC Í2 > INFORMATION FOR SEQ ID NO: 44: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 79 base pairs (B) TYPE: nucleic acid (C) CHAIN earrings (D) TOPOLOGY: linear (XI) DESCRIPTION OF THE SEQUENCE : SEQ ID NO: 44: CTTTCTTAGC GTCATCCTTC TTCGCAGCAT CCTTTTTCGC ATCGTCCGCA CTAAGCTTGA 60 TTTCCAGCTT GGTGCCTCC "79" (2) INFORMATION FOR SEQ ID NO: 45: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 70 base pairs ÍB) TYPE: nucleic acid (C) CHAIN simple (D) TOPOLOGY: linear (Xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 45: CTGCsAAGAA GGATGACGCT AAGAAAGACG ATGCTAAAAA GGACCTCGAG ATCCAGTTGG 60 TGCAGTCTGG 70 (2) INFORMATION FOR SEQ ID NO: 46: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 36 pairs of base (B) TYPE: nucleic acid (C) CHAIN eingle (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 46: GAGGAAGCTA GCTGAGGAGA CTGTGAGAGT GGTGCC 36 (2) INFORMATION FOR SEQ ID NO: 47: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 278 to inoasidoe (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (i) CHARACTERISTICS: (A) NAME / KEY: signal peptide pATKFLAG (peí B) (B) LOCATION: -22 a -1 ÍC) IDENTIFICATION PROCEDURE: hydrophobic (ix) CHARACTERISTICS: (A) NAME / KEY: mature protein of ecFv 9A4 »V - ^ - linkage-Vjj-segment FLAG (B) LOCATION: 1 to 256 (ix) FEATURES: (A) NAME / KEY: V ^ of 9A4 (B) LICALIZATION: 1 to 106 (ix) CHARACTERISTICS: (A) ) NAME / KEY: link 205C (B) L0CALI2ACI0N: 107 to 131 (ix) CHARACTERISTICS: (A) NAME / KEY: VH of 9A4 (B) LOCATION: 132 to 246 (ix) FEATURES: (A) NAME / KEY: FLAG segment (B) LOCATION: 249 to 256 ix) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 47: Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala -20 -15 -10 Ala Gln Pro Ala Met Ala Gln lie Val Leu Thr Gln Ser Pro Val Phe -5 1 5 10 Met Ser Ala Be Pro Gly Glu Lys Val Thr Met Thr Cys Ser Ala Ser 15 20 25 Ser Ser Val Ser Tyr Mee Tyr Trp Tyr Gln Gln Lys Pro sly Ser Ser 30 5 40 Pro Arg Leu Leu He His Wing Thr Ser Asn Leu Wing Ser Gly Val Pro 45 50 55 Val Arg Phe Ser Gly Gly Gly Ser Gly Thr Ser Tyr Ser Leu Thr He 60 65 70 Ser Arg Met Glu Wing Glu Asp Wing Wing Thr Tyr Tyr Cys G n Gln Trp 75 80 85 90 Arg Ser Tyr Thr Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu He He 95 100 105 Leu Ser Wing Asp Asp Wing Lys Lys Asp Wing Wing Lys Lys Asp Asp Wing 110 115 120 Lys Lys Asp Asp Wing Lys Lys Asp Leu Glu He Gln Leu Val sln Ser 125 130 135 Gly Pro Glu Leu Lys Lys Pro Gly Glu Thr Val Lys I Have Cys Lys 140 145 150 Wing Ser Gly Tyr Thr Phe Thr Asp Tyr Ser He His Trp Val Lys Gln 155 160 165 170 Wing Pro Gly Lys Gly Leu Lys Trp Met Gly Trp He Asn Thr Glu Thr 175 180 185 Gly Glu Pro Thr Tyr Wing Asp Asp Phe Lys Gly Arg Phe Wing Phe Ser 190 195 200 Leu Glu Thr Be Ala Be Thr Ala Tyr Leu Gln He Asn Asn Leu Lys 205 - 210 215 Asn Glu Asp Thr Wing Thr Tyr Phe Cys Wing Arg Gly Gly Ser Leu Asp 220 225 230 Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Wing Ser Asp Tyr 235 240 245 250 Lys Asp Asp Asp Asp Lys 255 (2) INFORMATION FOR SEQ ID NO: 43: (I) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 116 amino acids (B) TYPE: amino acid (D) linear TOPOLOGY ii) TYPE OF MOLECULE: protein (ix) CHARACTERISTICS : (A) NAME / KEY: mature protein VH of 5109 (B) LICALIZATION: 1 to 116 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 48: Glu Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Pro Gly Gly 5 10 15 Ser Leu Lys Leu Ser Cys Wing Wing Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Gly Met Ser Trp Val Arg Gln Thr Pro Asp Lys Arg Leu Glu Trp Val 35 40 45 Wing Thr He Asn Ser Asn Gly Gly Leu Thr Phe Tyr Wing Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr He Ser Arg Asp Asn Wing Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Arg Leu Lye Ser Gly Asp Ser Gly Met Tyr Tyr Cys 85 90 95 Val Arg Gly Tyr Ser Asn Tyr Wing Arg Trp Gly Gln Gly Wing Leu Val 100 105 lio Thr Val Ser Wing 115 (2) INFORMATION FOR SEQ ID NO: 49: (i) CHARACTERISTICS OF THE SEQUENCE: A) LENGTH 112 amino acids IB) TYPE: amino acid (D) linear TOPOLOGY (ii) TYPE OF MOLECULE: protein (i) CHARACTERISTICS: (A) ) NAME / KEY: mature protein V ^ of 5109 (B) LOCATION: * 1 to 112 xxi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 49: Asp Val Val Met Thr Gln Thr Pro Leu Thr Leu Ser Val Thr He Gly i 5 10"Gln Ser Ala Ser He Ser Cys Lys Ser Ser Gln Ser Leu Leu Gly Ser 2o 25 30 Asp Gly Leu Thr Tyr Leu He Trp Leu Leu Gln Arg Pro Gly Gln Ser 35 0 45 Pro Lys Arg Leu He Phe Leu Val Ser Glu Leu Asp Ser Gly Val Pro 50 55 60 Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys He 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Cys Gln Gly 85 90 95 Thr Hxs Phe Pro His Thr Phe Gly Wing Gly Thr Lys Leu Glu Leu Lys 100 105 HO (2) INFORMATION FOR SEQ ID NO: 50 (I) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid ÍC) STRING: simple (D) Linear TOPOLOGY (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 50: 24 GAAGTGCAGC TGGTGGAGTC TGGG (2) INFORMATION FOR SEQ ID NO: 51: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH 20 base pairs (B) TYPE: nucleic acid (C) CHAIN: simple (D) Linear TOPOLOGY (i) DESCRIPTION OF THE SEQUENCE: SEO ID NO: 51: GATGTTGTGA TGACCCAAAC 20 (2) INFORMATION FOR SEQ ID NO: 52: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (OR STRING: simple (D) TOPOLOGY: linear (i) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 52: CTGATCAGTC CAACTGTTCA GGAC 24 (2) INFORMATION FOR SEQ ID NO: 53: ii) SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 base pairs (B) TYPE: nucleic acid (OR STRING: simple (D) TOPOLOGY: linear (i) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 53: GTAVAACGAC GGCCAG 16 (2) INFORMATION FOR SEQ ID NO: 54: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 17 base pairs (B) TYPE: nucleic acid (OR STRING: simple (D) TOPOLOGY: linear (i) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 54: CAGGAAACAG CTATGAC 17 (2) INFORMATION FOR SEQ ID NO: 55: (i) CHARACTERISTICS OF THE SEQUENCE: 13B (A) LENGTH: 49 base pairs (B) TYPE: nucleic acid (OR STRING: linear (xi) DESCRIPTION OF SEQUENCE: SEO ID NO: 55: GAAGAGCGGC CCAGCCGGCC ATGGCCCAAG TGCAGCTGGT GGAGTCTGG 49 (2) INFORMATION FOR SEQ ID NO: 56: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 74 base pairs (B) TYPE: nucleic acid ÍC) CHAIN: simple (D) TOPOLOGY: linear (Xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 56; CGATCCGCCA CCGCCGCTGC CACCTCCGCC TGAACCGCCT CCACCACTCG AGACAGTOAC 60 CAOCGCCCCT TGGC 74 (2) INFORMATION FOR SEQ ID NO: 57: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: &6 base pairs (B) TYPE: nucleic acid (OR TOPOLOGY: linear (i) DESCRIPTION OF THE SEQUENCE : SEQ ID NO: 57: GGTTCAGGCG GAGGTGGCAG CGGCGGTGGC GGATCGTCTG ATGTTGTGAT GACCCAAACT 60 CCACTC 66 (2) INFORMATION FOR SEQ ID NO: 58: (i) SEQUENCE CHARACTERISTICS: ÍA) LENGTH: 87 base pairs ÍB) TYPE: nucleic acid (C) CHAIN: single (D) TOPOLOGY: linear (XI) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 58: GGAAGGAGCG GCCGCTTTCA GCTCCAGCTT GGTCCCAsCA CCCAACGTGT GAGGAAAATE 60 TGTACCTTGO GAGCAATAAT AAACTCC 87 (2) INFORMATION FOR SEQ ID NO: 59: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 36 base pairs (B) TYPE: nucleic acid (C) CHAIN: simple (D) TOPOLOGY: linear (Xi) ) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 59: GGAAGGAGCG GCCGCTTTCA GCTCCAGCTT GGTAGC 36 (2) INFORMATION FOR SEQ ID NO: 60: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) CHAIN: simple < D5 TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 60: CTCTTCTGAG ATGAGTTTTT G 21 (2) INFORMATION FOR SEQ ID NO: 61: (i) CHARACTERISTICS OF THE SEQUENCE: ÍA) LENGTH: 18 pairs of baeee ÍB) TYPE: nucleic acid (C) CHAIN: simple ÍD) TOPOLOGY: linear (Xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 61: GAGGCGGTTC AGGCGGAG 18 (2) INFORMATION FOR SEQ ID NO: 62: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) CHAIN: simple (D) TOPOLOGY: linear (i) ) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 62: GATCCGCCAC CGCCGCTG J-a (2) INFORMATION FOR SEQ ID NO: 63: (i) CHARACTERISTICS OF THE SEQUENCE: ÍA) LENGTH: 289 amino acids ÍB) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein iix) CHARACTERISTICS: ÍA ) NAME / KEY: signal peptide pCAN ß (B) LOCATION: -22 to -I (O IDENTIFICATION PROCEDURE: hydrophobic (ix) CHARACTERISTICS: (A) NAME / KEY: ripe protein scFv of 5109. V ^ -engar-V. ^ -marker segments <B) LICALIZATION: 1 to 267 (i) CHARACTERISTICS: (A) NAME / KEY: VH of 5109 ÍB) LICALIZATION: 1 to 116 (i) CHARACTERISTICS: (A) NAME / KEY: crimping (B) ) LOCATION: 117 to 132 (iX) CHARACTERISTICS: < A > NAME / KEY: V- of 5109 (B) LOCATION: 133 to 244 (i) CHARACTERISTICS: (A) NAME / KEY: segment myc (B) LOCATION: 248 to 257 (ix) FEATURES: (A) NAME / KEY: His segment (B) LOCATION: 262 to 267 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 63: Met Lys Lys Leu Leu Phe Ala lie Pro Leu Val Val Pro Phe Tyr Ala -20 -15 -10 Ala Gln Pro Ala Met Ala Glu Val Gln Leu Val Glu Ser Gly Gly Gly -S 1 5 10 Ser Val Gln Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala Wing Ser Gly 15 20 25 Phe Thr Phe Asn Thr Tyr Gly Met Ser Trp Val Arg Gln Thr Pro Asp 30 35 40 Lys Arg Leu Glu Trp Val Wing Thr lie Asn Ser Asn Gly Gly Leu Thr 45 50 55 Phe Tyr Wing Asp Ser Val Lys Gly Arg Phe Thr He Ser Arg Asp Asn 60 65 70 Wing Lys Asn Thr Leu Tyr Leu Gln Met Asn Arg Leu Lys Ser Gly Asp 75 80 85 90 Ser Gly Met Tyr Tyr Cys Val Arg Gly Tyr Ser Asn Tyr Wing Arg Trp 95 100 105 Gly Gln Gly Ala Leu Val Thr Val Ser Gly Gly Gly Gly Ser Gly 110 115 120 Gly Gly Gly Gly Gly Gly Gly Ser Ser Asp Val Val Met Thr Gln 125 130 135 Thr Pro Leu Thr Leu Ser Val Thr He Gly Gln Ser Wing Ser lie Ser 140 145 150 Cys Lys Ser Ser Gln Ser Leu Leu Gly Ser Asp Gly Leu Thr Tyr Leu 155 160 165 170 He Trp Leu Leu Gln Arg Pro Gly Gln Ser Pro Lys Arg Leu He Phe 175 180 185 Leu Val Ser Glu Leu Asp Ser Gly Val Pro Asp Arg Phe Thr Gly Ser 190 195 200 Gly Ser Gly Thr Asp Phe Thr Leu Lys He Ser Arg Glu Ala Glu wing 205 210 215 Asp Leu Gly Val Tyr Tyr Cys Cys Gln Gly Thr His Phe Pro His Thr 220 225 230 Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Ala Ala Ala Glu Gln Lys 235 240 245 250 Leu He Ser Glu Glu Asp Leu Asn Ala Ala Ala His His His His His 255 260 265 His (2) INFORMATION FOR SEQ ID NO: 64: ii) CHARACTERISTICS OF THE SEQUENCE; ÍA) LENGTH: 21 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (ix) CHARACTERISTICS: (A) NAME / KEY: modified and unusual amino acid (B) LOCALIZATION: 17 Í C) OTHER INFORMATION: "Xaa" is 4Hyp (X i) DESCRIPTION OF SEQUENCE: SEQ ID NO: 64: Glu Lys Gly Glu Pro Gly Asp Asp Ala Pro Ser Gly Ala Glu Gly Pro 10 15 Xaa Gly Pro Gln Gly 20 (2) INFORMATION FOR SEQ ID NO: 65: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 62 base pairs ÍB) TYPE: nucleic acid (C) CHAIN: simple (D) TOPOLOGY: linear (ix) CHARACTERISTICS: (A) NAME / KEY: branched nucleotides (B) LOCATION: 25 to 51 C) OTHER INFORMATION: rami isaaion level = 10% (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 65: GACTGTGAGA GTGGTGCCTT GGCCCCAGTA GTCAAGGCTA CCGCCCCTAG CACAGAAATA 60 TG 62 (2) INFORMATION FOR SEQ ID NO: 66: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 26 base pairs (B) TYPE: nucleic acid (C) CHAIN: single (D) TOPOLOGY: linear (xi) ) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 66: GCCAAGGCAC CACTCTCACA GTCTCC 26

Claims (14)

NOVELTY OF THE INVENTION CLAIMS

1. - A method for controlling protein fragments in a biological medium »comprising; digesting said biological medium with a capture antibody; said active capture antibody being against the sequences indicated in the Sequence List as SEQ ID NOS: 1 and 2; and contacting said biological medium with a detection antibody; said active capture antibody being against the sequences indicated in the Sequence List as SEQ ID NOS: 3 and 4; and detecting the amount of collagen fragments attached to dishwaste antitumor and dementing antibodies; or contacting said biological medium with a capture antibody; said capture antibody being active against the sequences indicated in the Sequence List as SEQ ID NOS: 3 and 4; and to put in half biological medium disho are an antithesis of detest; wherein the active de-active antisense antibody shows the sequences indicated in the Sequence List co or SEQ ID NOS: 1 and 2 and detects the amount of collagen fragments bound to said detection capture antibodies.

2. A method of claim 1 wherein said protein fragments are fragments of collagen generated by the excision of the articular cartilage by collagenase.

3. - A method of claim 2 »wherein said protein fragments are generated by the cleavage of type II collagen by the collagenase.

4. A method for controlling protein fragments in a biological medium comprising; contacting said biological medium with an active antibody against the sequences indicated in the Sequence Listing as SEQ ID NOS: 3 and 4; and detecting the amount of collagen fragments bound to said antibody.

5. A method of claim 4 wherein said protein fragments are collagen fragments.

6. A procedure of claim 1 wherein the capturing and detesting antibodies are monoslonal antibodies.

7. A method of claim 2 wherein said detection and detection antibodies are monoclonal antibodies.

8. A method of claim 1 wherein said capture antibody is an antibody obtained by genetic engineering.

9. A method of claim 1 wherein said detection antibody is an antibody obtained by genetic engineering.

10. A method of claim 1. wherein said sapure antibody. called 9A4. has the VH sequence indicated in the Sequence List as SEQ ID NOS: 5 and 32 and the sequence of V- ^ Indicated in the Sequence List as SEQ ID NOS: 6 and 33.

11. A procedure of claim 1 »Wherein said detection antibody, designated 5109. has the sequence of H indicated in the Sequence List as SEQ ID. NOS: 10 and 48 and the sequence of ^ indicated in the List of Sequences as SEQ ID NOS: 11 and 49.

12. A method of claim 4. wherein said antibody is a monoclonal antibody.

13. A method of claim 4. wherein said antibody is an antibody obtained by genetic engineering.

14. A method of claim 4. wherein said anti-body "denominated 5109. has the sense of V, -. indicated in the Sequence List as SEQ ID NOS: 10 and 48 and the sequence of V ^ indicated in the Sequence List as SEC ID NOS: 11 and 49. 15.- The antibody, called 9A4. having the sequence of V ^ indicated in the Sequence List as SEQ ID NOS: 5 and 32 and the sequence of - ^ Indicated in the Sequence List as SEQ ID NOS: 6 and 33. 16.- The antibody. termed 5109. having the sequence of Vj, indicated in the Sequence List as SEQ ID NOS: ÍO and 4B and the sequence of V ^ indicated in the Sequence List as SEQ ID NOS: 11 and 49. 17.- One line cell that produces a specific binding molecule that binds to peptides that consist esensially of the structures indicated in the Sequence List as SEQ ID NOS: 1 or 2. having the cell line the identification characteristics of ATCC HB 12436. 18.- A selular line that produces a specific binding molecule that binds to peptides which consist essentially of the structures indicated in the List of Sesuensias as SEQ ID NOS: 3 or 4. having the line seal the identification sarasteristisas of ATCC HB 12435. 19.- A form obtained by genetic engineering of the antibody 9A4. such as that expressed by E. coli P9A4ICAT7-1 or P9A4IF-5, which has the characteristics of ATCC-98593 and ATCC-98592. respectively. 20. A form obtained by genetic engineering of the anti- body 5109 »such as that which comes from E. coli p5109CscFv7, which can replace 5109» and which has the characteristics of ATCC-98594. 21. A bispecific antibody produced by hybridization of antibodies 9A4 and 5109, in which half of the antibody resonates with its respective binding molecule. 22.- A bispecific antibody that is a genetically engineered combination of antibodies 9A4 and 5109 »produced by co-biosion of the domains - ^ and Vj-. of the two antibodies in the form V ^ 510 510) -linker-VH (5109) -linker-Vt, (9A4) -engaged-Vflí A4) and equivalents thereof. 23. A method for controlling fragments of cabbage in a biological medium "comprising: contacting said biological medium with a bispecific antibody 9A4 / 5109 of claim 22. and detecting the amount of collagen fragments bound to said antibody. 24.- A bispecific antibody of the claim 22 produced from genetically modified variants of antibodies 5109 and 9A4. 25.- An antibody related to antibodies 9A4 or 5109 by virtue of the VL and Vj genes, which come from the genes of the same germ line as the corresponding genes V ^ and VH of 9A4 and 5109 and where changes in the secuensia. but the sockets V - ^ - V--. maintain the binding properties of antibody 9A4 or 5109. 26.- An antibody of claim 1 which is labeled to facilitate detection. 27. An antibody of claim 26, wherein said marker is radioactive, "optical," enzymatic, polarizing, fluoressing, or in vivo-enhancing. 28. An antibody of claim 1 which is labeled to facilitate the capture of said antibody. 29. An antibody of claim 28 »wherein said marker is biotin or magnetic particles.