Antigenic gm-csf peptides and antibodies to gm-csf

SUMMARY OF THE INVENTION

In a first aspect, the present invention relates to a human recombinantly expressed monoclonal antibody, which antibody specifically binds to GM-CSF and comprises a heavy chain comprising the CDR1 region of sequence No. 40, the CDR2 region of sequence No. 41 and the CDR3 region of the sequence No. 42, and a light chain comprising the CDR1 region of sequence No. 43, the CDR2 region of sequence No. 44 and the CDR3 region of sequence No. 45, wherein the binding affinity of the subject antibody to human GM-CSF has a dissociation constant (Kd) of less than 1 χ 10 "^ten M. In a second aspect, the present invention relates to a preparation comprising antibodies of the invention and a pharmaceutically acceptable carrier.

In a third aspect, the present invention relates to an antibody or a preparation of the invention intended for use in the treatment of an inflammatory disease, malignancy or a GM-CSF mediated infectious disease.

SHORT DESCRIPTION OF CHILDREN

The following figures refer to different antibodies. For the resolution of doubts, the antibody of the invention is an antibody that is included in the claims. Other antibodies referred to in the description of the figures are presented for comparative purposes and do not form part of the invention.

Figure 1 shows an ELISA antigen panel for the selection of antigen-specific human monoclonal antibodies. In response to human GM-CSF, three GM-CSF-specific human monoclonal antibodies (E5, G7 and E10) and none of the other antigens contained in the panel entered. Antibody 215 is a mouse monoclonal antibody that binds to human GMCSF (hGM-CSF) and mouse GM-CSF (mGM-CSF).

[0023] Figures 2A and 2B illustrate the high specificity of human monoclonal antibodies to native human GM-CSF. As shown in Figure 2A, the soluble human GM-CSF was bound to mouse hybridoma cells having on their surface immunoglobulins directed against human GM-CSF. The monoclonal antibody E5 was then added to the reaction mixture, and its binding to human GM-CSF was quantified using goat anti-human immunoglobulin coupled to FITC. The E5 antibody did not bind to any of the surface proteins expressed in mouse hybridoma cells (middle panel), binding only to soluble GM-CSF bound to cell surface immunoglobulins (bottom panel). As shown in Figure 2B, human GM-CSF labeled with phycoerythrin (PE), or PE-GM,

The binding of PE-GM was competed with the excess of unmarked GM-CSF (bottom panel).

[0024] Figure 3 shows the secretion of antigen-binding immunoglobulins

IgG by hybridoma cells after class switching. E5 (parental) hybridoma cells were subjected to the interventions described in "Materials and Methods". Hybridoma clones in which class switching took place (switched clones) were identified using the ELISPOT method. To assess the IgM or IgG binding, an ELISA was carried out to quantify the specific binding to human GM-CSF applied to the plates. Monoclonal IgG antibodies resulting from class switching showed a comparable binding to antigen as in the case of parental IgM.

[0025] Fig. 4 shows the GM-CSF-mediated inhibition of the growths of the TF-1 cell line by fully human monoclonal antibodies. The human-GM-CSF dependent human TF-1 erythroid cell line (ATCC, Virginia, USA) was propagated in complete RPMI1640 medium containing recombinant human GM-CSF at 10 ng / ml (PeproTech, New Jersey, USA). On the day before the experiment, TF-1 cells were grown in 0.1% FBS in the absence of GM-CSF. The starved TF-1 cells were harvested and rinsed twice with test medium (simple RPMI medium with 0.5% BSA). Cells were resuspended in assay medium and plated into 96-well microplates at a concentration of 10,000 cells / well. The wells contained either test medium, GM-CSF in an amount of 100 pg / ml, or GM-CSF pre-incubated for one hour with test antibodies or antibodies for isotype control at the concentrations indicated in the figure. After 3 days, 40 μl of Cell Titer reagent (Promega, Wisconsin, USA) was added to each well and the plates were further incubated at 37 ° C for one hour. An optical density measurement (OD) was performed at 490 nm in a spectrophotometer, subtracting the medium background from all samples. The percentage of GM-CSF neutralization was calculated from the following formula: subtracting the nutrient background from all samples. The percentage of GM-CSF neutralization was calculated from the following formula: subtracting the nutrient background from all samples. The percentage of GM-CSF neutralization was calculated from the following formula:

100 - (OD from Ig / OD without Ig) χ 100.

[0026] Figure 5A shows the results of the ELISA test, showing that all tested E5-3D2 subclones secrete high concentrations of immunoglobulins. The E5-3D2 hybridoma line was propagatedfor 60 generations, and then the production stability was evaluated by analyzing the frequency of cell production. Random subclones (X1-X10) obtained from 3D2 cells by limiting dilution were selected and a quantitative evaluation of immunoglobulin production was performed using an ELISA based assay. Absorbance at 405 nm was normalized to colony size by visual evaluation of wells containing cells. Fig. 5B shows that the specific productivity determined during the logarithmic growth phase was pg / cell / day after inoculation of 3D2 cells in a bioreactor with an agitator containing 1 liter of serum-free medium, production of immunoglobulins and registration of viable cells in the period from 1 to 5 days.

[0027] Fig. 6A shows the results of an exemplary deletion of a single nucleotide in a BAT marker found in E5 hybridoma cells treated with inhibitors of mismatched bases. Dotted lines crossing the middle histogram peak represent the size of the wild type (wt) or truncated fragment (-1 nt).

According to Figure 6B, parental cells and cells that inhibited the repair of mismatched bases were seeded into microplates, resulting in 3763 and 2437 immunoglobulin secreting clones (OD> 0.2), respectively. Immunoglobulin concentrations were determined by ELISA, and the frequency of clones with an optical density greater than 1 was recorded, expressing as a percentage of the total number of screened clones.

[0028] Figure 7 is a diagram describing the individual steps of obtaining a fully human hybridoma cell line.

[0029] Fig. 8 panel and anti-gene / dose antibody ELISA and antigen-specific human monoclonal antibodies. Human GM-CSF reacted with three GM-CSF-specific human monoclonal antibodies - E10, G9 and E5 (not described) - and none of the other antigens contained in the panel. To increase the sensitivity of the test, efficient antigen coating was optimized using specific monoclonal antibodies against various antigens. Antibodies 615 and 215 are mouse monoclonal antibodies directed against human GM-CSF.

[0030] Figure 9 shows the isotype fixing method of the monoclonal antibody E10. To determine the isotype of E10 antibodies, a standard analysis was performed using anti-human antibodies that were specific for IgG, IgG1, IgG2, IgG3, IgM, Lk and LA Fc.

[0031] Carthage 10 is presented with anaiysis using the Weseern Hot przeciwkaiza method!

monoclonal E10 and G9. Antibodies E10 and G9 were analyzed by western blotting to determine if they cross-react with human recombinant GM-CSF. The mAb215 antibody is a mouse anti-human GM-CSF neutralizing monoclonal antibody serving as a positive control. A lysate of tumor cells was applied to lane 1 as a negative protein control. Track No. 2 was loaded with 500 ng of rhGM-CSF (PeproTech, New Jersey, USA). As shown in Figure 5, antibodies E10 and G9 have reacted with human GM-CSF.

[0032] Figure 11 shows BIACORE antibody analysis of antibody E10. Antibody

E10.20 was characterized by an association constant (ka) of 2.47 χ 104 dissociation constant (kd) of 2.16 χ 10 "^fiveand an overall affinity (Kd) of 0.87 nM.

[0033] Figure 12 shows a method for determining the isotype of monoclonal G9. In order to determine the isotype of G9 antibodies, a standard analysis was performed using anti-human antibodies with specificity for IgG, IgG1, IgG2, IgG3, IgM, Lk and LA Fc (in duplicate samples, Figure 3).

[0034] Figure 13 shows a BIACORE analysis of the G9 antibody. Antibody

10G9.1 was characterized by an association constant (ka) of 8.47 χ 10⁶, a dissociation constant (kd) of 9.27 χ 10 "^fiveand an overall affinity (Kd) of 0.87 nM.

[0035] Figure 14 shows a map of the G9 epitope.

The overlapping peptides used to map the G9 epitope binding site correspond to amino acid sequences 35-38 and 64-85.

[0036] Figure 15 shows the mapping analysis of the G9 epitope carried out by western blot. The overlapping peptides overlap the human GM-CSF protein sequence, corresponding to amino acid sequences 35-38 and 65-86. Peptides reactive with the antibody include sequences No. 35-38.

DETAILED DESCRIPTION OF EMBODIMENTS [0037] Various terms associated with particular aspects of the present invention are used in the text of the specifications and claims. Unless otherwise indicated, these terms should be understood in accordance with their generally accepted meaning in the art. Other terms, namely those defined in this document, should be interpreted in accordance with the definitions given herein.

[0038] It should be understood that the present invention is not limited to specific reagents, compounds, preparations, or biological systems that may, of course, be varied. It is also to be understood that the terminology used herein is intended to describe only specific embodiments and is not intended to be exhaustive. The nouns used in this specification and in the claims attached thereto also refer to their plural counterparts, unless it is clear from the context that they do not. The term "cell" therefore also refers, in total, to two or more cells, etc.

[0039] Each subset of all combinations and sub-combinations of ranges as well as specific numbers included in these ranges are cited within the scope of the invention.

[0040] As used in this document οΕΐ'θέ! Θηίθ "(^ ł ^ ooD" in οόηίθ5ίθηίη for rational values, e.g. quantity, duration, etc., includes deviations from the specified value of ± 20% or ± 10%, more preferably ± 5 %, more preferably ± 1%, and even more preferably ± 0.1% - according to which of them are suitable for implementing the disclosed methods.

[0041] Under the term "disease" is meant "between me", a pathogen infection - a virus, a bacterium, a fungus or a parasite. Examples of viruses include, but are not limited to, Severe Acute Respiratory Syndrome (SARS), hepatitis B virus or hepatitis C virus, influenza virus, varicella virus, adenovirus , herpes simplex virus or herpes simplex virus type, rinderpest virus, rhinovirus, echovirus, rotavirus, respiratory syncytial virus, papilloma virus, papovavirus, cytomegalovirus, echinovirus, arbovirus, hantavirus, Coxsackie virus, mumps virus, measles virus, rubella virus, polyovirus, as well as human immunodeficiency virus (HIV) human immunodeficiency virus) type I or type II. Examples of bacteria are, among others, Ebola, Staphylococcus AE, Plasmodium (malaria), M. tuberculosis, mycobacteria, mycoplasmas, Neisseria and Legionella. Examples of parasites are: rickettsiae and chlamydia.

[0042] Under the influence of "inflammatory diseases", it is understood to be the snare of "mire" and chronic immunologic and autoimmune diseases, for example rheumatoid arthritis, autoimmune diseases, inflammatory diseases of the kidneys and diseases. inflammatory lungs, for example bronchial asthma and chronic obstructive pulmonary disease (COPD), and multiple sclerosis and autoimmune encephalomyelitis.

[0043] Under the condition that the disease is derived from and affected by the own tissues of the subject and directed against them, or coseregulation or manifestation of the same disease, a 5θΗθΓζθηϊθ disease is understood; or disorder or condition resulting therefrom. Examples of autoimmune diseases include arthritis (rheumatoid arthritis, e.g. acute arthritis, chronic arthritis, gout or gouty arthritis, acute gouty arthritis, acute immune arthritis, chronic inflammatory arthritis, degenerative arthritis, collagen type II arthritis, infectious arthritis, borreliosis arthritis, proliferative arthritis, psoriatic arthritis, Still's disease, spondylitis and juvenile rheumatoid arthritis,

[0044] The term "conservatively modified variants" refers to both the amino acid sequence and the nucleic acid sequence. For specific nucleic acid sequences, conservatively modified variants are understood to mean nucleic acids encoding identical or substantially identical amino acid sequences or, in the case of a nucleic acid not encoding any amino acid sequence, substantially identical sequences. Due to the degeneracy of the genetic code, any protein is encoded by a large number of functionally identical nucleic acids. The amino acid alanine, for example, is encoded by any of the following codons: GCA, GCC, GCG and GCU. So in every position in which the alanine is determined by a given codon, this codon can be replaced with any of the other codons listed above without affecting the encoded polypeptide. These varieties of nucleic acids are called "silent varieties" and are one of the subtypes of conservatively modified variants. Each nucleic acid sequence encoding a polypeptide mentioned herein also describes every possible mute variant of said nucleic acid. It will be clear to the person skilled in the art that any codon in a nucleic acid (with the exception of AUG, which is usually the only codon encoding methionine, as well as TGG, which is usually the sole codon encoding tryptophan) can be modified to obtain a functionally identical molecule. Accordingly, it is assumed that any silent nucleic acid coding for a polypeptide is included in each of the sequences described with respect to the expression product,

[0045] The adjective "recombinant" when used in reference to, for example, a cell, nucleic acid, protein or vector, means that a given cell, a given nucleic acid, a given protein or a given vector has been modified by introducing heterologous nucleic acid or the protein either by altering the native nucleic acid or protein or means that the given cell is derived from the cell in the above modified manner. Thus, for example, recombinant cells express genes that are not present in the native (non-recombinant) form of these cells or express native genes that undergo abnormal or reduced expression in native cells or do not undergo them at all.

[0046] The term "acid ηηΗθίηον" "t" poly-adjuic sequence "refers to a single- or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5 'to the end of 3'. Nucleic acids may also include modified nucleotides that allow the polymerase to be read correctly by the chain and do not affect the expression of the polypeptide encoded by the nucleic acid. This also applies, for example, to conservatively modified variants.

the addition or deletion of a single amino acid or a small percentage of amino acids in the coding sequence results in a conservatively modified variant thereof if this change results in the amino acid being replaced by a chemically similar amino acid. Conservative substitution tables with functionally similar amino acids are well known in the art.

Such conservatively modified variants exist in the present invention along with polymorphic variants, interspecific homologies and alleles, and are not mutually exclusive. Each of the eight groups listed below contains the amino acids constituting their mutual conservative substitutes: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); 8) cysteine ​​(C), methionine (M) (33). The term "conservative substitution" further includes the use of a substituted amino acid in place of the non-extracted parent amino acid, provided that the polypeptide of interest also has the required binding activity.

The term & quot; amino acid & quot; refers to naphthaline strands and synthetic amino acids as well as to amino acid analogs and amino acid mimics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are amino acids that are encoded by the genetic code, as well as those that are later modified, for example, hydroxyproline, γ-carboxyglutamate and O-phosphoserine. The term "amino acid analogue" refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e. an α-carbon which is bonded to hydrogen, a carboxyl group, an amino group and an R group, e.g. homoserine, norleucine, sulfoxide methionine, methionine methylsulfonate. These analogs have modified R groups (e.g. norleucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. The term "amino acid mimetic" refers to a chemical compound whose structure differs from the general chemical structure of the amino acid, but which functions similar to a naturally occurring amino acid.

[0049] For the determination of amino acid molecules, their commonly known three letter symbols or single letter characters recommended by the IUPAC-IUB Commission for Biochemical Nomenclature (see table 1 below) are used. The commonly accepted single-letter codes are used to write the nucleotide names.

<td colspan="3">TABLE 1</td>

<td>SYMBOL</td><td></td><td></td>

<td>1-letter</td><td>3-letter</td><td>AMINO ACID</td>

<td>Y</td><td>Tyr</td><td>L-tyrosine</td>

<td>G</td><td>Gly</td><td>L-glycine</td>

<td>F</td><td>phe</td><td>L-phenylalanine</td>

<td>M</td><td>Underworld</td><td>L-methionine</td>

<td>AND</td><td>ala</td><td>L-alanine</td>

<td>S</td><td>Cheese</td><td>L-serine</td>

<td>AND</td><td>How much</td><td>L-isoleucine</td>

<td>The</td><td>Lion</td><td>L-leucine</td>

<td>T</td><td>Thr</td><td>L-threonine</td>

<td>V</td><td>hr</td><td>L-valine</td>

<td>P</td><td>Pro</td><td>L-proline</td>

<td>K</td><td>list</td><td>L-lysine</td>

<td>H</td><td>His</td><td>L-histidine</td>

<td>Q</td><td>Gln</td><td>L-glutamine</td>

<td>IS</td><td>Glu</td><td>L-glutamic acid</td>

<td>IN</td><td>Trp</td><td>L-tryptophan</td>

<td>R</td><td>Angry</td><td>L-arginine</td>

<td>D</td><td>Asp</td><td>L-aspartic acid</td>

<td>N</td><td>own</td><td>L-asparagine</td>

<td>C</td><td>Cys</td><td>L-cysteine</td>

[0050] It should be noted that all the amino acid sequences are filed here with the formulas whose left-to-right orientation corresponds to the generally accepted direction from the amino terminus to the carboxy terminus.

[0051] The term "in vitro" or "ex" used in the minor document refers to the artificial environment and to processes or reactions occurring in an artificial environment, which may be, inter alia, test-tubes and tissue cultures. to the natural environment (for example an animal or a cell) and to processes or reactions occurring in the environment.

[0052] Specify "pharmacistically-acceptable wave"

and any grammatical variations of these terms are used interchangeably when referring to formulations, carriers, diluents and reagents, and mean that these materials can be administered into or onto the surface of the human body without causing undesirable physiological effects to attain an intolerance to the administration of the preparation.

[0053] The term "pharmaceutical vehicle admission" refers to reagents, excipients, cells, compounds, materials, preparations and / or pharmaceutical forms which, in terms of sound medical judgment, are suitable for use in contact with human tissues. and animals without causing excessive toxic effects, irritancy, allergic reactions or other complications, at a reasonable benefit / risk ratio. According to a more detailed description provided herein, pharmaceutically acceptable carriers suitable for use in the present invention include gases, liquids as well as semisolid and solid materials.

[0054] The words "patient" and "patient" are used interchangeably and refer to mammals, including humans and non-human primates, as well as to experimental animals, including rabbits, dogs, cats, rats, mice. Accordingly, the terms "patient" and "patient" herein mean any mammal to which the formulations of the invention may be administered. In certain embodiments of the present invention, the patient will suffer from an infectious or inflammatory disease and / or an autoimmune disease. In some embodiments of the present invention, the patient will have a malignant tumor.

Eligible screening methods are used to determine patients eligible for antibody or formulation treatment in order to determine the severity of a given disease or condition in a patient or risk factors for a disease or condition that is suspected or a target for the action of said antibody or disease. preparation. Said screening methods include, for example, diagnostic tests to determine whether a given patient has an infectious disease, an inflammatory disease, a malignant tumor or an autoimmune disease. The abovementioned and other routine methods allow the clinician to select patients who need treatment.

[0055] The term "treatment" refers to any indication of success in treating or ameliorating a course of, for example, an infectious disease, an inflammatory disease, inter alia, an inflammatory disease mediated by GM-CSF, a malignant tumor or an autoimmune disease. The term includes, but is not limited to, any objective or subjective parameter, e.g. resolution of the disease; remission; reducing the severity of symptoms or making the condition more tolerable to the patient; deceleration of the rate of degenerative or functional deterioration; or making the last stage of the degenerative process become less physically harmful for the patient. Treatment or alleviation of symptoms may be based on objective or subjective parameters, including the results of a diagnostic test. Accordingly, the term "treatment" includes administering the compounds or agents of the present invention to delay, alleviate or arrest or inhibit the development of symptoms or conditions associated with malignancy, an infectious disease, an inflammatory disease, including an inflammatory disease mediated by GM-CSF, or an autoimmune disease. Treatment includes, for example, inhibiting the growth of dysplastic cells, inhibiting the progression of malignant tumor or neoplastic disease, maintaining inhibition of tumor growth, and inducing remission. including an inflammatory disease mediated by GM-CSF, or an autoimmune disease. Treatment includes, for example, inhibiting the growth of dysplastic cells, inhibiting the progression of malignant tumor or neoplastic disease, maintaining inhibition of tumor growth, and inducing remission. including an inflammatory disease mediated by GM-CSF, or an autoimmune disease. Treatment includes, for example, inhibiting the growth of dysplastic cells, inhibiting the progression of malignant tumor or neoplastic disease, maintaining inhibition of tumor growth, and inducing remission.

[0056] The term "therapeutic compound" as used herein refers to a compound useful in the prophylaxis or treatment of a disease or condition, e.g. malignancy, an infectious disease, an inflammatory disease or an autoimmune disease.

[0057] "toxin" refers to or eliminates the disease, symptoms or side effects of the disease in a patient or the prevention thereof. The term "effective amount" refers to the amount needed to elicit the desired effect. A "therapeutically effective amount" means an amount that when administered to a patient as a treatment for a disease, condition or disorder will be sufficient to effect the treatment of the disease.

[0058] "Co-administration" or "concurrent administration" as used herein includes administration of active agents (e.g., monoclonal antibodies, chemotherapeutic agents, biomolecules) together or in combination, together, or one of them prior to or after administration of the other. of them. The agents administered simultaneously may be administered in the same or different ways, simultaneously or sequentially, provided that they are administered in a manner that ensures all of the administered agents achieve effective concentrations at the site of action. One of ordinary skill in the art will have no difficulty in determining the appropriate time of administration, the order of administration and dosage for the individual drugs and formulations of the present invention.

[0059] The term "donor cells" is used herein in a broad sense to refer to cells fused to human B lymphocytes to obtain hybridomas. These cells include, but are not limited to, those found in rodents in the sense understood by those skilled in the art, rodent cell lines, human cell lines, and avian cell lines. Cell lines can be obtained by any methods known to those skilled in the art.

what is necessary for performing various functions, such as binding for complement. There are five classes of immunoglobulins, whereby the primary structure of the heavy chain in the Fc region is decisive for belonging to a given class. Specifically: alpha chains correspond to class A (IgA) immunoglobulins, delta chains - class D immunoglobulins (IgD), epsilon chains - class E immunoglobulins (IgE), gamma chains - class G immunoglobulins (IgGs), and m-chains - class M immunoglobulins (IgM). As used herein, the terms "immunoglobulin" or "antibody" include all sub classes of alpha, delta, epsilon, gamma and mi classes and refer to any natural (e.g., IgA and IgM) and synthetic multimers of a four chain immunoglobulin structure. Antibodies covalently, specifically and reversibly bind antigen.

[0061] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies. This means that the individual antibodies composing the said population are identical except for any naturally occurring mutations that may be present in small amounts. Monoclonal antibodies may, for example, be produced by a single clone of antibody-producing cells. In contrast to polyclonal antibodies, monoclonal antibodies are monospecific (e.g. they have specificity for one epitope on one antigen). The "monoclonal" adjunct points to an antibody trait meaning obtaining it from a largely homogeneous antibody population and should not be interpreted as indicating the need to produce this antibody by any particular method. For example, monoclonal antibodies that would be used in accordance with the present invention can be produced in a hybridoma method first described by Kohler et al. In Nature 1975, 256: 495 or by recombinant DNA methods. Monoclonal antibodies may also, for example, be isolated from phage libraries using the techniques described by Marx et al. In The Journal of Molecular Biology 1991, 222: 581-597. may be produced by hybridoma first described by Kohler et al. in Nature 1975, 256: 495 or by recombinant DNA methods. Monoclonal antibodies may also, for example, be isolated from phage libraries using the techniques described by Marx et al. In The Journal of Molecular Biology 1991, 222: 581-597. may be produced by hybridoma first described by Kohler et al. in Nature 1975, 256: 495 or by recombinant DNA methods. Monoclonal antibodies may also, for example, be isolated from phage libraries using the techniques described by Marx et al. In The Journal of Molecular Biology 1991, 222: 581-597.

[0062] The term "chimerized" as used herein refers to an immunoglobulin in which the variable regions of the heavy and light chains are not of human origin, while the constant regions of the heavy and light chains are.

[0063] The term "humanized" refers to an immunoglobulin, such as an antibody in which the amino acids directly involved in antigen binding - forming complementarity determining regions, i.e. the CDRs (complementarity determining regions) - in the heavy and light chains are not of human origin, while the remainder of the immunoglobulin molecule, that is, the framework regions of the variable heavy and light chain parts, and the heavy and light chain constant regions are of human origin.

[0064] A "fully human" refers to a molecule and an antibody in which the whole molecule is of human origin or is composed of an amino acid sequence identical to the human form of the antibody.

The term "conformational epitope" as used herein refers to the determinηΓηηηο ίοζ "antigenic determinant serving as the antibody binding site. antigen other than an uninterrupted series of amino acids.

[0066] A "hybridoma" refers to a "cellular" product that has grown between a cultured tumor lymphocyte and a B or T lymphocyte subjected to pioneering (priming) and which product exhibits a specific cell immune potential. parental.

[0067] (n) "GM-CSF" refers to and suggests growth factors with a glycoprotein structure that control the production, differentiation and function of granulocytes and monocyte-macrophages. An example, though not the only one, of this kind is described in U.S. Patent No. 5 602 007 (34).

[0068] As used in this document, οΕΐ'θέ! Θηίθ "mole-particle" refers to any molecule that can be conjugated with an antibody of the invention, co-administered with an antibody of the invention, administered before or after administration of the antibody of the invention or otherwise associated with the antibody of the invention. Biomolecules include, among others, enzymes, proteins, peptides, amino acids, nucleic acids, lipids, carbohydrates and their fragments, homologs, analogs, derivatives and combinations thereof. Examples of biomolecules are interleukin-2, interferon alpha, interferon beta, interferon gamma, Rituxan, Zevalin, Herceptin, Erbitux and Avastin. Biomolecules may be native, recombinant or synthetic molecules and may be the result of modifying the native form by, for example, glycosylation, acetylation,

[0069] The polypeptides of the present invention can be synthesized from amino acids using techniques known to those skilled in the art of polypeptides. Generally, these methods include sequential addition to the lengthening peptide chain of one or more amino acid residues or suitably protected amino acid residues. Typically, the amino or carboxyl group of the first amino acid residue is protected using a suitable selectively removable protecting group. In the case of amino acids containing a reactive side group (e.g. lysine), another selectively removable protecting group is used.

[0070] Various methods for obtaining the polypeptides of the invention are known in the art (patents with numbers WO 89/06657, WO 92/22315 and WO 98/49191, US patents with numbers 5 260 273, 5 164 369,

407 914, 5 789 381, 5 952 303, 6 013 619, 6 013 764, 6 120 795 and 6 613 734).

[0071] According to the present invention, additional residues may be added to one or the other end of the polypeptide, for example, to form a "linker", by means of which the polypeptide may conveniently be attached to a tag or solid matrix or carrier. Labels, solid templates and vehicles that can be used with the polypeptides of the present invention are known in the art, and some examples thereof are also described herein.

[0072] Amino acid linkers are usually composed of at least one amino acid residue and may be 40 residues long or longer, though more often their length ranges from 1 to 10 residues. Typical amino acid residues used to form linkers are tyrosine, cysteine, lysine, glutamic acid and aspartic acid, and the like. The polypeptide sequence of the present invention may also differ from the natural sequence by modifying the acylation of the terminal NH 2 group, e.g. acetylation, or by thioglycolic acid amidation or amidation of the terminal carboxyl group, e.g. with ammonia, methylamine etc.

[0073] Although many useful polypeptides, for example polypeptides with sequences No. 1-5, are indicated herein, it is also true that many other molecules - including naturally occurring, though not very commonly, amino acids, metabolites and catabolites of natural amino acids, substituted amino acids and analogs of amino acids, as well as amino acids of configuration D - find use in the molecules and compositions of the present invention. In addition, "designed" amino acid derivatives, analogs and mimetics as well as polymers, including frameworks made of non-amide linkers, can also be used in the various compounds and compositions of the present invention.

[0074] As used herein, the terms "analogs" and "derivatives" of polypeptides and amino acid residues are intended to include amino acid metabolites and catabolites, as well as molecules comprising linkers, backbones, side chains or side groups that differ from those typically encountered in compounds referred to as "naturally occurring" forms of L-amino acids (The terms "analog" and "derivative" may conveniently be used interchangeably herein). Within the meaning of the terms "analog" and "derivative" used in this document, therefore, there are also D-amino acids, molecules mimicking amino acids, as well as amino acids with "designed" side chains (i.e. those that can replace one or more amino acids in the surfactant molecule) ).

[0075] For example, in addition to those listed in Table 1, also amino acid metabolites such as homoarginine, citrulline, ornithine and α-aminobutanoic acid are useful in the molecules and compositions of the present invention.

[0076] In another variation, one would like to form a molecule with a more "rigid" conformation, and one way to achieve this would be to add a methyl or other group to the α-carbon in the given amino acid.

[0077] Furthermore, substituted amino acids, which are generally not derived from proteins, although they occur naturally and are useful as disclosed herein, include the following examples: L-canavanine; 1-methyl-L-histidine; 3-methyl-L-histidine; 2 methyl-L-histidine; α, ε-diaminopimelic acid (L-form, meso-form or both forms); sarcosine; L-ornitynobetainę; Histidine betaine (herzynina); L-citrulline; L-fosfoargininę; Doctopine; o-carbamyl-D-serine; γ-aminobutanoic acid; and also β-lysine. The following D-amino acids and D-amino acid analogs, including those listed below, are also useful in the proteins, polypeptides and compositions of the present invention: D-alanine, Dserin, D-valine, D-leucine, D-isoleucine, D-alloisolamine, D- phenylalanine, D-glutamic acid, D-proline and D-allohydroxyproline and the like.

[0078] Amino acids may include analogs, metabolites, catabolites and derivatives regardless of the time and place of modification. In principle, modified amino acids can be placed in three categories: 1) catabolites and amino acid metabolites; 2) amino acids modified as a result of post-translational modifications (for example, side chain modifications) and 3) amino acids modified in non-metabolic or non-catabolic processes (for example, the synthesis of modified amino acids or derivatives in the laboratory).

[0079] A chain of amino acids or residual amino acids including longer or truncated side chains can be easily designed by adding or subtracting methylene groups in chain (linear or branched) or ring (hydrocarbon or heterocyclic) chain systems. Said linear and branched chain structures may also contain non-carbon atoms, e.g. S, O or N. Useful components of the surface-active molecules herein may also be fatty acids. The above-mentioned designed side chains can be terminated with moieties containing (R ') groups having a charge or polar groups or no (R) groups.

[0080] Analogs are also useful, including molecules obtained by using different linkers. Also useful as disclosed herein are molecules with side chains joined by linkers other than amide linkers, e.g. molecules comprising amino acid side chains or other side chains (R- or R'-), whose components are linked by combinations, e.g. carboxylic acid. or phosphoro ether, ethylene, methylene, ketone or ether. In general, any amino acid side chain or molecule containing an R or R 'group may be useful as disclosed herein.

[0081] The mullions according to the present invention may rustle a dimer attached by a suitable linker, for example peptide dimers joined by cysteine ​​molecules. (As any professional in the field knows, two cysteine ​​molecules can be combined with each other by a disulfide bond due to the oxidation of their thiol groups). Such linkers, i.e. bridges, may thus lead to cross-linking of various polypeptide chains, dimers, trimers, etc. Other suitable linkers that can be used to link together peptide dimers and / or other peptide multimers include those listed above, e.g. combinations carboxy or phospho ester, ethylene, methylene, ketone or ether, etc.

[0082]

It will be apparent to a person skilled in the art that a variety of modifications may be made to individual amino acids, linkers and / or the chain itself, which modifications will lead to the molecules within the scope of the present invention as long as these molecules exhibit biological activity (e.g. antigenic) as described in this document.

[0085] Previously reported methods for obtaining hybridomas secreting human monoclonal antibodies have been used (patent WO 2004/046330) using primary human B lymphocytes. Peripheral blood mononuclear cells (PBMCs), preferably human PBMCs, are immunized ex vivo in the presence of a target antigen, and then immortalized by fusion with donor cells, or selected PBMCs whose sera show high immunoreactivity to the antigen of interest are identified.

[0086] Hybrid cells obtained from donor cells are screened for the secretion of monoclonal antibodies having specificity for the target antigen. In some embodiments, methods are provided for producing hybridoma cells producing monoclonal antibodies directed against a target antigen from immunized ex vivo immunoglobulin producing cells, which methods include: a) a combination of peripheral blood mononuclear cells consisting of cells that produce immunoglobulin with an ex vivo target antigen; b) converting the cells producing immunoglobulins into donor cells, resulting in hybridoma cells; c) determining the antigen binding by antibodies produced by said hybridoma cells, and then d) selecting hybridoma cells that produce antibodies binding to the target antigen, thereby producing hybridoma cells producing antibodies directed against the target antigen. PBMCs can come from healthy donors. The target antigen is preferably GM-CSF, and more preferably the antigen consists of one of the amino acid sequences 35-38.

[0087] Methods for obtaining hybridomas that produce monoclonal antibodies directed against the target antigen associated with the disease may also consist of the following steps: a) ex vivo fusion of peripheral blood mononuclear cells consisting of B lymphocytes that produce immunoglobulins from a patient with a given disease or from a donor subjected to exposure to antigen - with donor cells, resulting in hybridoma cells; b) determining the target antigen binding by antibodies produced by said hybridoma cells; and then c) selecting hybridoma cells that produce antibodies binding to the target antigen, thereby obtaining hybridoma cells that express the antibodies directed against the target disease. preferably, The target antigen is an antigen associated with a disease that is preferably a malignant tumor, an infectious disease or an autoimmune disease. More preferably, the disease-associated antigen is GM-CSF. The target antigen is preferably GM-CSF, and more preferably the antigen consists of one of the amino acid sequences 35-38. In one preferred embodiment of the invention, the subject exposed to the antigen has been exposed to GM-CSF or may have pulmonary proteinosis (PAP).

The present invention provides methods for the preparation of hybridoma cells producing antibodies directed against a target antigen (which is GM-CSF or antigenic peptides of that factor) from immunized ex vivo immunoglobulin producing cells, which methods are based on: a) a combination of peripheral blood mononuclear cells consisting of from an immunoglobulin producing cell with an ex vivo target antigen; b) converting the cells producing immunoglobulins into donor cells, resulting in hybridoma cells; c) performing screening for binding of the target antigen by immunoglobulins produced by said hybridoma cells, thereby obtaining hybridoma cells producing antibodies directed against the target antigen. PBMCs can come from healthy donors. The target antigen may consist of an antigenic polypeptide. The target antigen preferably consists of one of the amino acid sequences 1-5, more preferably one of the sequence No. 3-5.

[0089] The present invention also provides methods of making hybridoma cells producing antibodies directed against a target antigen (which is, for example, GM-CSF or antigenic peptides of that agent), which methods are based on: a) selecting peripheral blood mononuclear cells consisting of cells producing immunoglobulins from a donor exposed to antigen; b) performing the fusion of immunoglobulin producing cells with myeloma cells, resulting in hybridoma cells; c) performing screening for binding of the target antigen by immunoglobulins produced by said hybridoma cells, thereby obtaining hybridoma cells producing antibodies directed against the target antigen. The target antigen may consist of an antigen polypeptide of the invention. The target antigen preferably consists of one of the amino acid sequences 1-5, more preferably one of the sequence No. 3-5. The donor could have been exposed to the expon the GM-CSF or antigen peptides of this factor, or it may have pulmonary proteinosis (PAP).

[0090] Donor cells (e.g., myeloma cells) may express a protein inhibitor of mismatched base repair. Hybridoma cells may express a protein inhibitor repair of mismatched bases. A protein inhibitor of mismatch repair can be introduced into hybridoma cells after myeloma fusion with immunoglobulin producing cells. A protein inhibitor of the mismatch repair can be introduced into a donor or myeloma cell prior to fusion with immunoglobulin producing cells. Donor or myeloma cells or antibody-producing cells may show a natural deficiency in the mechanism of repair of mismatched bases.

[0091] Spam repair mismatches of mismatched bases include the dominant negative allele of the mismatch repair genes. The dominant negative alleles of the mismatch repair genes include, but are not limited to, dominant negative alleles of the PMS2, PMS1, PMSR3, PMSR2, PMSR6, MLH1, GTBP, MSH3, MSH2, MLH3 or MSH1 genes and mutL and mutS gene homologs. Polypeptides that can interfere with the repair of mismatched bases can also be used. For example, the dominant negative allele of the mutL gene PMS2 contains the first 133 amino acids of PMS2. Further determination of the amino acids in the mutL homologs reveals amino acids

LSTAVKELVENSLDAGATNIDLKLKDYGVDLIEVSDNGCGVEEENFE (SEQ ID NO: 6) and

LRQVLSNLLDNAIKYTPEGGEITVSLERDGDHLEITVEDNGPGIPEEDLE (sequence No. 7) or fragments thereof. Protein inhibitors of mismatched base peptides thus include Sequence No. 6 and 7 polypeptides and fragments thereof. A protein inhibitor of repairing mismatched bases may be inactivated. A protein inhibitor of the mismatch repair can, for example, be inactivated before or after the identification of a hybridoma cell producing monoclonal antibodies against the target antigen. Inactivation of the protein inhibitor repair of mismatched bases can be achieved by any method known in the art, for example, by removing an inducer from a cell or a protein inhibitor repairing mismatched bases (resulting in the deprivation of the protein inhibitor inhibitor of mismatched bases in the cell).

[0092] In some methods of obtaining hybridoma cells, hybridoma cells are exposed to a chemical inhibitor of repair of mismatched bases. Chemical mismatch repair inhibitors used in some embodiments of said methods include, but are not limited to, at least one anthracene, ATPase inhibitor, nuclease inhibitor, RNA interference molecule, polymerase inhibitor, and antisense oligonucleotide specifically hybridizing to the nucleotide encoding the mismatch repair protein (patent WO 2004/046330). The chemical inhibitor mentioned may be an anthracene compound with the following formula:

<img file="PL1981909T3_D0001.tif" />

an organosilicone compound or a carbohydrate optionally containing one or more alkylated hydroxyl groups; wherein said heteroalkyl, heteroaryl and substituted heteroaryl include at least one heteroatom which is oxygen, sulfur, metal, phosphorus, silicon or nitrogen; and wherein the substituent of the above-mentioned substituted alkyl group, substituted alkenyl group, substituted alkynyl group, substituted aryl group and substituted heteroaryl group is halogen, CN, NO2, lower alkyl alkyn, aryl, heteroaryl, aralkyl, aralkoxy, guanidino, alkoxycarbonyl group, alkoxy, hydroxyl, carboxyl and amino; and wherein said amino groups are optionally substituted with an acyl group or, in numbers from 1 to 3, with an aryl group or an alkyl group of lower alkynes. In some embodiments, R 5 and R 6 are hydrogen. In other embodiments, R1 to R10 is independently hydrogen, hydroxy, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, phenyl, tolyl, hydroxymethyl, hydroxypropyl or hydroxybutyl. Examples of the above-mentioned anthracene, inter alia, 1,2-dimetyloantracen, 9,10-dimetyloantracen, 7,8-dimetyloantracen, 9,10difenyloantracen, 9.10 dihydroksymetyloantracen-9-hydroxymethyl-10-metyloantracen, dimetyloantraceno-1,2- diol, 9-hydroxymethyl-10-methylanthracene-1,2-diol, 9-hydroxymethyl-10-methylanthracene-3,4-diol and 9,10-di-m-tolylanthracene. hydroxymethyl, hydroxypropyl or hydroxybutyl. Examples of the above-mentioned anthracene, inter alia, 1,2-dimetyloantracen, 9,10-dimetyloantracen, 7,8-dimetyloantracen, 9,10difenyloantracen, 9.10 dihydroksymetyloantracen-9-hydroxymethyl-10-metyloantracen, dimetyloantraceno-1,2- diol, 9-hydroxymethyl-10-methylanthracene-1,2-diol, 9-hydroxymethyl-10-methylanthracene-3,4-diol and 9,10-di-m-tolylanthracene. hydroxymethyl, hydroxypropyl or hydroxybutyl. Examples of the above-mentioned anthracene, inter alia, 1,2-dimetyloantracen, 9,10-dimetyloantracen, 7,8-dimetyloantracen, 9,10difenyloantracen, 9.10 dihydroksymetyloantracen-9-hydroxymethyl-10-metyloantracen, dimetyloantraceno-1,2- diol, 9-hydroxymethyl-10-methylanthracene-1,2-diol, 9-hydroxymethyl-10-methylanthracene-3,4-diol and 9,10-di-m-tolylanthracene.

[0093] The inhibitory chemical mentioned above can be introduced into the growth medium of cells. The chemical inhibitor mentioned above can be removed from the hyper-mutant hybridoma cells to restabilize the genome of these cells. This method may also involve inactivation of a chemical inhibitor to repair mismatched bases, thus stabilizing the hyper-mutated hybridoma gene.

[0094] These methods may also consist in cloning genes responsible for the production of immunoglobulins from antibody-producing cells and transfecting a mammalian expression cell with immunoglobulin genes, wherein said genes are operably linked to expression control sequences.

[0095] The invention also provides methods for the maintenance of mammalian expression cells producing high affinity antibodies to the target antigen from ex vivo immunized immunoglobulin producing cells, which methods include: a) a combination of peripheral blood mononuclear cells consisting of cells that produce immunoglobulins with the target antigen ex vivo b) converting the cells producing immunoglobulins into donor cells, resulting in hybridoma cells; c) determining the antigen binding by antibodies produced by said hybridoma cells,

d) zotptn: to lyophilize the repair of mismatched bases or to clone the immunoglobulin genes from the hybridoma to the mammalian expression cell, wherein the repair of mismatched bases in the mammalian expression cell is inhibited, and then e) selection of hybridoma cells that produce antibodies binding to the target antigen, thereby obtaining hybridoma cells producing antibodies directed against the target antigen. Alternatively to the steps a) and b), ex vivo fusion of peripheral blood mononuclear cells may be used consisting of immunoglobulin producing B cells derived from a patient with a given disease or from a donor exposed to the antigen - with donor cells, resulting in hybridoma cells .

[0096] Pre-mycotaxin &lt;4ο5Ρηγοζ3 is also the method of oryvivan of mammalian expression cells that produce high affinity antibodies to the target antigen from ex vivo immunized cells that produce: a) a combination of peripheral blood mononuclear cells consisting of cells that produce immunoglobulins with the ex vivo target antigen; b) performing a fusion of immunoglobulin producing cells with myeloma cells, resulting in hybridoma cells; c) performing screening for binding of the target antigen by antibodies produced by said hybridoma;

[0097] Disclosed herein is an excess method for the preparation of mammalian expression cells that produce high titers of high affinity antibodies from immunized ex vivo immunoglobulin producing cells, which method involves: a) a combination of peripheral blood mononuclear cells consisting of cells that produce immunoglobulin with the target antigen ex vivo b) converting the cells producing immunoglobulins into donor cells, resulting in hybridoma cells; c) determining the antigen binding by antibodies produced by hybridoma cells; d) cloning the immunoglobulin genes from the hybridoma into a parent mammalian expression cell, wherein the repair of mismatched bases in the mammalian expression cell is inhibited; e) incubating the mammalian parent cell or hybrid expression cell to allow for mutagenesis, thus obtaining the hyper-mutant mammalian expression cells; f) selection of hyper-mutant mammalian expression cells secreting antibodies with a higher affinity for the target antigen than antibodies produced from parent hybridoma cells or selection of hyper-mutant mammalian expression cells secreting higher antibody titers than mammalian parental expression cells, thereby obtaining mammalian expression cells producing antibodies directed against the antigen target from immunized ex vivo cells producing immunoglobulins.

[0098] In the document, moreover, the οθ ^ η ^ νν3ηί3 method of mammalian expression cells expressing high titres of high affinity antibodies from immunized ex vivo immunoglobulin producing cells, which method consists in: a) a combination of peripheral blood mononuclear cells consisting of cells that produce immunoglobulins with an ex vivo target antigen; b) performing a fusion of immunoglobulin producing cells with myeloma cells, resulting in hybridoma cells; c) performing screening for antigen binding by antibodies produced by said hybridoma; d) cloning of the immunoglobulin genes from the hybridoma to a parent mammalian expression cell, wherein the repair of mismatched bases in the mammalian expression cell is inhibited, or optimizing the preparation of the hybridoma by inhibiting repair of mismatched bases; e) incubating the mammalian parent cell or hybridoma expression cell to allow mutagenesis, thus obtaining the hyper-mutant mammalian expression cells; f) screening hyper-mutant mammalian expression cells secreting antibodies with a higher affinity for the target antigen than antibodies produced from hybridoma cells, and then g) screening for hyper-mutant mammalian expression cells secreting higher antibody titers than mammalian parental expression cells, thereby obtaining mammalian expression cells that produce high titers of high affinity antibodies from immunized ex vivo immunoglobulin producing cells.

[0099] In some embodiments, the antibody methods are screened using an ELISA-based assay or other assays that allow for quantitative antibody-antigen binding - known in the art. Crowther, JR (2001) The ELISA Guidebook, 1st ed. Humana Press, Totowa, NJ.

[0100] Said assays may allow screening for hybridized hybridomas that produce antibodies with higher affinity than antibodies produced by parent hybridomas.

[0101] The method of the invention may further comprise selecting selected hyper-mutant antibody-producing cells that produce higher antibody titers than the originally selected cells.

[0102] Methods of immunoglobulin-producing cells and cells with myeloma cells and myeloma cells useful in these methods are also known in the art (Kohler &amp; Milstein, "Derivation of specific tissue and tissue culture by Fusion", European Immunology 1976, 6 : 511-9).

[0103] Human B lymphocytes used to obtain monoclonal antibodies intended for human administration may be a potential carrier for viral transmission. Fusion partner cells and peripheral blood mononuclear cells from donors can be pre-screened to confirm the absence of viral DNA, for example by PCR, including DNA belonging to Type 1 and 2 immune deficient viruses, hepatitis B and C viruses, cytomegalovirus, and herpervirus type 6 and Epstein-Barr virus.

[0105] The invention also includes antibodies directed against target antigens produced by hybridoma cells prepared by the methods disclosed herein. Antibodies of the invention further include antibodies obtained by recombinant methods using the polynucleotides of the invention. Antibodies of the invention are fully human monoclonal antibodies.

[0106] Preferred antibodies of the invention specifically bind to an epitope, for example a conformational epitope, of a target antigen. Antibodies of the invention are preferably directed against the antigen associated with the disease, which may be, for example, GM-CSF.

Antibody-producing cells were deposited with the ATCC (10801 University Blvd., Manassas, Virginia 20110-2209, USA) on January 18, 2007 (10G9) and February 2, 2007 (E1O) and assigned the catalog numbers of the PTA-8173, respectively. and PTA-8193. Examples of anti-GM-CSF antibodies of the invention are antibodies produced by these cells.

[0107] It will be clear to a person skilled in the art that the specificity of the antibodies is primarily determined by the six CDRs, and in particular by the heavy chain CDR3 region (Kala M., The Journal of Biochemistry 2002, 132: 535-41; Morea V. et al., The Journal of Molecular Biology 1998, 275: 269-94; Chothia C. et al., The Journal of Molecular Biology 1987, 196: 901-17). However, antibody framework regions may play a role in antigen-antibody interactions (Panka DJ et al.

Proceedings of the National Academy of Sciences of the United States of America 1988, 85: 30804), especially in terms of affecting the conformation of the CDR loop (Foote J. et al., The Journal of Molecular Biology 1992, 224: 487-99) . Antibodies of the invention may thus comprise any combination of CDR or FWR regions of heavy or light chains exhibiting specificity for GM-CSF. In order to obtain GM-CSF specific binding antibodies in accordance with the specifications that have been set forth and exemplified herein, domain shuffling experiments that are routinely performed in the art may be used (Jirholt P. et al., Gene 1998, 215: 471-6, Soderlind E. et al. Nature Biotechnology 2000, 18: 852-6).

[0110] It should be understood that due to the natural sequence variation with high probability existing between heavy and light chains and between genes coding for these chains, a person skilled in the art will expect a certain level of variation within or within the genes that encode them, wherein the unique binding properties (e.g., specificity and affinity) of the antibodies of the present invention will still be preserved. This expectation is partly due to the degeneracy of the genetic code, and partly to the known success of the evolutionary variability of conserved amino acid sequences, which does not significantly alter the nature of the encoded protein. Accordingly, such variants and homologs are considered substantially identical and are within the scope of the present invention.

[0111] Antibodies of the invention thus include variants with single or multiple substitutions, deletions, additions or exchanges of amino acids while maintaining the biological properties (e.g., binding affinity and immunoreactivity) of the antibodies of the invention. The skilled person is able to obtain variants with single or multiple substitutions, deletions, additions or exchanges of amino acids. These variants include, but are not limited to: a) variants in which at least one amino acid residue has been substituted by a conservative or non-conservative amino acid, b) variants in which at least one amino acid has been added to or removed from the polypeptide, (c) variants in which the least one amino acid has a substituent, d) variants in which the polypeptide has been linked to another peptide or polypeptide, for example, a fusion partner, a protein tag or other chemical moiety capable of conferring useful attributes to the polypeptide, e.g. an antibody epitope, a polyhistidine sequence, a biotin group. Antibodies of the invention may include variants in which the amino acid residues derived from one species are substituted for the corresponding residues from another species, in conserved or non-conserved positions. In other embodiments, the amino acid residues at non-conserved positions are substituted with conservative or non-conservative residues. Techniques for obtaining these variants, including genetic techniques (suppressions, deletions, mutations, etc.), chemical and enzymatic, are known to those of ordinary skill in the art. a protein tag or other chemical moiety capable of conferring useful attributes to the polypeptide, e.g. an antibody epitope, a poly-histidine sequence, a biotin group. Antibodies of the invention may include variants in which the amino acid residues derived from one species are substituted for the corresponding residues from another species, in conserved or non-conserved positions. In other embodiments, the amino acid residues at non-conserved positions are substituted with conservative or non-conservative residues. Techniques for obtaining these variants, including genetic techniques (suppressions, deletions, mutations, etc.), chemical and enzymatic, are known to those of ordinary skill in the art. a protein tag or other chemical moiety capable of conferring useful attributes to the polypeptide, e.g. an antibody epitope, a poly-histidine sequence, a biotin group. Antibodies of the invention may include variants in which the amino acid residues derived from one species are substituted for the corresponding residues from another species, in conserved or non-conserved positions. In other embodiments, the amino acid residues at non-conserved positions are substituted with conservative or non-conservative residues. Techniques for obtaining these variants, including genetic techniques (suppressions, deletions, mutations, etc.), chemical and enzymatic, are known to those of ordinary skill in the art. poly-histidine sequence, biotin group. Antibodies of the invention may include variants in which the amino acid residues derived from one species are substituted for the corresponding residues from another species, in conserved or non-conserved positions. In other embodiments, the amino acid residues at non-conserved positions are substituted with conservative or non-conservative residues. Techniques for obtaining these variants, including genetic techniques (suppressions, deletions, mutations, etc.), chemical and enzymatic, are known to those of ordinary skill in the art. poly-histidine sequence, biotin group. Antibodies of the invention may include variants in which the amino acid residues derived from one species are substituted for the corresponding residues from another species, in conserved or non-conserved positions. In other embodiments, the amino acid residues at non-conserved positions are substituted with conservative or non-conservative residues. Techniques for obtaining these variants, including genetic techniques (suppressions, deletions, mutations, etc.), chemical and enzymatic, are known to those of ordinary skill in the art. in conserved or non-conserved positions. In other embodiments, the amino acid residues at non-conserved positions are substituted with conservative or non-conservative residues. Techniques for obtaining these variants, including genetic techniques (suppressions, deletions, mutations, etc.), chemical and enzymatic, are known to those of ordinary skill in the art. in conserved or non-conserved positions. In other embodiments, the amino acid residues at non-conserved positions are substituted with conservative or non-conservative residues. Techniques for obtaining these variants, including genetic techniques (suppressions, deletions, mutations, etc.), chemical and enzymatic, are known to those of ordinary skill in the art.

[0112] The skilled person in the art will be clear that in some cases the pairing of a given heavy chain (H chain) with different light chains (L chains) or pairing of a given L chain with different H chains will lead to the same or greater antibodies. specificity and / or the same or greater affinity than the native combination. The present invention is not therefore limited to the preferred combinations of H and L chain pairs, and the antibodies of the present invention comprise various combinations of H and L chain pairs - including, but not limited to, H and L chains described herein, or other H and L chains known the person skilled in the art, or combinations of which, in any other manner, have been experimentally determined,

[0113] The preferred antibodyUU according to the invention is made up of two heavy chains. Preferred antibodies of the invention consist of two light chains. Antibodies consisting of two heavy chains and two light chains according to the invention are more preferred.

[0114] Antibody VEGAG substitutes variants with single or multiple substitutions, deletions, additions or exchanges of amino acids while maintaining the biological properties (e.g., binding affinity and immunoreactivity) of the antibodies of the invention. The skilled person is able to obtain variants with single or multiple substitutions, deletions, additions or exchanges of amino acids. These variants include, but are not limited to: a) variants in which at least one amino acid residue has been substituted by a conservative or non-conservative amino acid, b) variants in which at least one amino acid has been added to or removed from the polypeptide, (c) variants in which the least one amino acid has a substituent, d) variants in which the polypeptide has been linked to another peptide or polypeptide, for example, a fusion partner, a protein tag or other chemical moiety capable of conferring useful attributes to the polypeptide, e.g. an antibody epitope, a polyhistidine sequence, a biotin group. Antibodies of the invention may include variants in which the amino acid residues derived from one species are substituted for the corresponding residues from another species, in conserved or non-conserved positions. In another embodiment, the amino acid residues at the non-conserved positions are substituted with conservative or non-conservative residues. Techniques for obtaining these variants, including genetic techniques (suppressions, deletions, mutations, etc.), chemical and enzymatic, are known to those of ordinary skill in the art. Antibodies of the invention also include antibody fragments.

[0115] Antibodies of the invention have Target Antigen affinity characterized by a dissociation constant (Kd) of less than 10 "^ten. In one embodiment, K d is smaller than 1 χ 10-ii. In some embodiments, the Kd is smaller than 1 χ 10-7. In other embodiments, Kd is less than 1 χ 10-7. In further other embodiments, Kd is less than 1 χ 10-14 In still other embodiments, Kd is less than 1 χ 10-15 [0116] Antibodies of the invention include derivatives that have been modified, for example by covalent attachment of any kind of molecule to antibodies that do not prevent the antibody from binding to its epitope. Examples of suitable derivatives include, but are not limited to, glycosylated antibodies and fragments, acetylated antibodies and fragments, pegylated antibodies and fragments, phosphorylated antibodies and fragments, as well as amidated antibodies and fragments. Antibodies of the invention may themselves be derivatized by known protecting / blocking groups, proteolytic breakdown, binding to cellular ligand or other proteins and the like. Antibodies of the invention may further comprise at least one non-classical amino acid as described above. In some embodiments of the invention, the GM-CSF or its epitopes are coupled to a protein having immunogenicity to increase the immunogenicity of the antigen. Said immunogenicity protein can be any protein enhancing the immune response of cells, e.g., tetanus toxoid C (TT), keyhole limpet haemocyanin, albumin, ovalbumin, chick albumin (CAB), albumin hemocyanin (keyhole limpet haemocyanin). , bovine serum albumin, thyroglobulin, diphtheria toxoid, BCG, cholera toxin, etc.

[0117] Antibodies of the invention may have post-translational moieties that increase the activity of the antibody or its persistence. These moieties include sulfur, methyl group, carbohydrate, phosphorus and other chemical groups commonly found in immunoglobulin molecules.

[0118] Antibodies according to the invention may belong to any isotype, wherein the antibody isotype may be altered as a result of in vivo class switching or the use of genetic engineering methods.

[0119] Disclosed herein are nucleotide sequences encoding polypeptides of the invention. The nucleic acids of the invention include, but are not limited to, genomic DNA, DNA, cDNA, RNA, bi- and single-stranded nucleic acids, and complementary sequences thereof.

[0122] In the process, we will make polynecotides in the base of the invention (and the peptides encoded by them) include a leader sequence. Any leader sequence known in the art can be used. The leader sequence may include, but is not limited to, a restriction site and / or a translation start site.

[0123] Disclosed herein are expression vectors composed of polynucleotides encoding antibodies of the invention and host cells, including recombinant host cells that express said polynucleotides.

[0124] Recombinant expression vectors that occupy the sequence encoding the polypeptide of interest are also provided. The expression vector may contain one or more additional sequences, including regulatory sequences (e.g. promoter, enhancer), selection marker and polyadenylation signal.

[0125] Sculpted expression devices of synthetic, genomic or cDNA-derived nucleic acid fragments encoding at least one recombinant protein capable of being functionally associated with appropriate regulatory elements. Said regulatory elements may include a transcriptional promoter, sequences encoding suitable mRNA ribosome binding sites, and sequences controlling the termination of transcription and translation. Expression vectors, particularly mammalian expression vectors, may also include one or more non-transcriptional elements, e.g. an origin of replication, a suitable promoter and enhancer associated with the gene to be expressed, other 5'- or 3'-flanking sequences that are not transcribed, 5'- or 3 ' -translational sequences that are non-translated (e.g., ribosome binding sites required), polyadenylation site, donor and acceptor splice sites, or transcription termination sequences. An origin of replication can also be built into the expression vector, by which the vector acquires the ability to replicate in the host organism.

[0126] The sequences controlling transcription and translation in expression vectors intended for use to effect transformation of vertebrate cells can be derived from viral sources. Exemplary vectors can be prepared as described by Okayama and Berg, Molecular and Cellular Biology 1983, 3: 280.

[0127] Selection markers that can be used in this system include selectable markers known in the art, and can be positive and negative selectable markers, including antibiotic resistance genes (e.g., neomycin resistance gene, hygromycin resistance gene, gene) resistance to kanamycin, tetracycline resistance gene, penicillin resistance gene), HSV-TK, HSV-TK derivatives for ganciclovir selection, or bacterial gene for purine nucleotide phosphorylase for 6-methylpurine selection (Gadi et al., Gene Therapy 2000, 7: 1738-1743). [0034] The nucleic acid sequence encoding the selectable marker or its cloning site may be upstream or downstream of the nucleic acid sequence encoding the polypeptide of interest or its cloning site.

[0128] The vector may include one or more promoters, including a constitutive, inducible, host-specific and / or tissue-specific promoter. For example, commonly used promoters and enhancers are derived from human cytomegalovirus (CMV), type 2 adenovirus, simian virus 40 (SV40) and polyomavirus. Viral genomic promoters and control and / or signal sequences may be used to stimulate and maintain expression, depending on the compatibility of the host cells. Depending on the type of cells in which the vector is to be expressed, promoters derived from the genes of basic metabolism (for example promoters of the β-globin gene, thymidine kinase and EF1α) may also be used.

[0129] The vectors may contain one or more internal ribosome binding sites, i.e. an IRES (internal ribosome entry site) sequence. Incorporation of IRES sequences into fusion vectors may favorably affect the expression of certain proteins.

[0130] Such a vector system may comprise one or more polyadenylation sites (as is the case, for example, in the case of SV40 virus), which may be located in the upstream or downstream direction relative to any of the above-mentioned acid sequences. nucleic.

[0131] The components can be connected to each other in a continuous manner. They can also be arranged in a way that ensures optimal spatial separation for the expression of gene products, which is achieved, for example, by introducing spacer nucleotides between individual open reading frames (ORFs). These components can also be arranged in a different way. Regulatory elements, for example an IRES motif, can also be arranged to provide optimal spatial separation for expression.

[0132] Conjugates may be screened for expression and may be selected under positive selection conditions and / or screened for recombinant protein expression. Cells expressing the recombinant protein are expanded and screened for subclones exhibiting the desired phenotype.

[0133] Cells, eukaryotic and pookaryotic eukaryotes can be transformed using expression vectors. Thus, a host cell has been described herein that has been transformed using an expression vector. Suitable host cells are preferably eukaryotic cells, more preferably plant cells, rodent cells or human cells, e.g. NSO, CHO, perC.6, Tk-ts13, BHK, HEK293, COS-7, T98G, CV-1 / EBNA, L cells , C127, 3T3, HeLa, NS1, Sp2 / 0 myeloma cells and BHK cell lines.

[0134] In general, the transfection will be carried out using a cell suspension or a single cell, although other methods may also be used as long as a sufficient fraction of cells or tissues subjected to transfection incorporate the subject polynucleotide, thus allowing the multiplication and use of transfected cells. Transfection techniques are well known. Several transformation protocols are known in the art, see, for example, Kaufman, Methods in Enzymology 1988, 185: 537. It will be clear to a person skilled in the art that which transformation protocol will be suitable will be determined by the type of host cell and the nature of the gene of interest. The basic elements of any transformation protocol include the introduction of a nucleic acid sequence encoding a protein of interest into a suitable host cell, followed by the identification and isolation of host cells that have incorporated vector DNA in a manner that ensures sustained expression. Techniques for introducing polynucleotides include, but are not limited to, electroporation, transduction, cell fusion, use of calcium chloride, and packaging of polynucleotides with a lipid to fuse the cell of interest. If the transfection is stable, i.e. if the selection gene (marker gene) is expressed at a stable level for many generations, then a cell line is formed. and then identification and isolation of host cells that embedded vector DNA in a way that ensures sustained expression. Techniques for introducing polynucleotides include, but are not limited to, electroporation, transduction, cell fusion, use of calcium chloride, and packaging of polynucleotides with a lipid to fuse the cell of interest. If the transfection is stable, i.e. if the selection gene (marker gene) is expressed at a stable level for many generations, then a cell line is formed. and then identification and isolation of host cells that embedded vector DNA in a way that ensures sustained expression. Techniques for introducing polynucleotides include, but are not limited to, electroporation, transduction, cell fusion, use of calcium chloride, and packaging of polynucleotides with a lipid to fuse the cell of interest. If the transfection is stable, i.e. if the selection gene (marker gene) is expressed at a stable level for many generations, then a cell line is formed. use of calcium chloride and packaging of polynucleotides with lipid to fuse with the cell of interest. If the transfection is stable, i.e. if the selection gene (marker gene) is expressed at a stable level for many generations, then a cell line is formed. use of calcium chloride and packaging of polynucleotides with lipid to fuse with the cell of interest. If the transfection is stable, i.e. if the selection gene (marker gene) is expressed at a stable level for many generations, then a cell line is formed.

[0135] A common method of roasting, particularly of mammalian cells, is the calcium phosphate precipitation method. Another method is the fusion of bacterial protoplasts with mammalian cells by polyethylene glycol (PEG, polyethylene glycol) (Schaffner et al., Proceedings of the National Academy of Sciences of the United States of America 198, 77: 2163). Yet another method is electroporation, which may further be used to introduce DNA directly into the cytoplasm of the host cell in the manner described, for example, by Potter et al., Proceedings of the National Academy of Sciences of the United States of America 1988, 81: 7161 .

[0136] TThnogenesis can also be carried out using polyliposomal reagents, for example lipofectin and lipofectamine (Gibco BRL, Gaithersburg, Maryland, USA), which form lipid complexes with nucleic acid (in the form of liposomes) and which complexes, when added to cultured cells, facilitate the uptake of nucleic acid by the cell.

[0137] Once the cells have been identified, demonstrating protein expression, it can be expanded and selected. Transfected cells can be selected in a variety of ways, e.g. for the expression of the polypeptide of interest. Cells in which the vector also contains an antibiotic resistance gene can be selected for antibiotic resistance, resulting in a positive selection for vector-containing cells. These cells can be left to propagate under selective conditions.

[0138] Once the clone producing the given protein is identified, the cell line can then be screened to identify subclones exhibiting one or more desired phenotypes, including high titer expressing cells, improved growth properties and / or ability to produce proteins having the desired biochemical characteristics, for example, as a result of protein modification and / or changed post-translational modifications. These phenotypes may result from the intrinsic properties of a given subclone or may be the result of mutagenesis. Mutagenesis can be caused by chemical compounds, ultraviolet radiation, ionizing radiation, viruses, insertional mutagens, defective DNA repair or a combination of these methods.

[0141] In prophylactic applications, the pharmaceutical preparations are administered to a patient susceptible to chtrtba or a subcutaneous state or otherwise at risk of developing a disease or condition (for diseases or conditions herein, e.g. malignant tumor, infectious disease or inflammatory disease) in a prophylactically effective amount . People at risk include, but are not limited to, people with a family history of malignant disease, infectious disease or inflammatory disease, people who have been previously treated for malignant disease, infectious disease or inflammatory disease, and people with any other clinical features indicative of an increased probability of developing malignant tumor, infectious disease or inflammatory disease. In other words, an endangered person is any person who has been recognized that it is exposed to a greater risk of developing malignant tumor, an infectious disease or an inflammatory disease than in the general population. The term "prophylactically effective amount" should be understood as the amount of a pharmaceutical preparation that has the effect of preventing the development or recurrence of a malignant tumor, an infectious disease or an inflammatory disease. The amounts of prophylactic effectiveness of a given pharmaceutical preparation are usually determined on the basis of their effect compared to the effect observed when a second pharmaceutical preparation lacking the active ingredient is administered to a person in a similar situation. The term "prophylactically effective amount" should be understood as the amount of a pharmaceutical preparation that has the effect of preventing the development or recurrence of a malignant tumor, an infectious disease or an inflammatory disease. The amounts of prophylactic effectiveness of a given pharmaceutical preparation are usually determined on the basis of their effect compared to the effect observed when a second pharmaceutical preparation lacking the active ingredient is administered to a person in a similar situation. The term "prophylactically effective amount" should be understood as the amount of a pharmaceutical preparation that has the effect of preventing the development or recurrence of a malignant tumor, an infectious disease or an inflammatory disease. The amounts of prophylactic effectiveness of a given pharmaceutical preparation are usually determined on the basis of their effect compared to the effect observed when a second pharmaceutical preparation lacking the active ingredient is administered to a person in a similar situation.

[0142] In therapeutic settings, the formulations are administered to a patient suspected or diagnosed with the disease in a therapeutically effective amount to cure or at least partially arrest the symptoms of the disease (biochemical and / or histological), including its complications and intermediate phenotypes pathological in the course of the development of the disease.

[0143] Both in profliacious, as well as occasional drug regimens, these agents are usually administered in several doses to obtain a sufficient response. In typical cases, the response is monitored and the dose is repeated if the response starts to weaken.

[0144] In the case of a monoclonal anthelm, the effective dose amounts for the treatment of diseases, e.g. malignant tumors, infectious diseases or inflammatory diseases, as described herein, depend on a variety of factors including the mode of administration, the target site, the condition the physiological patient, whether the patient is a human or animal, other medications being given, and whether these antibodies are administered for prophylactic or curative purposes. Usually the patient is a human, although other mammals may also be treated.

[0145] Dosage depends on the severity and responsiveness of the disease state to be treated, wherein the treatment may last from several days to several months either to cure or to a lesser extent of the disease state. The optimal dosing regimen can be determined by calculation based on measurements of drug accumulation in the patient's body. A person of average skill in the field is able to easily determine the optimal doses, the optimal mode of administration and optimal intervals between successive doses. Optimal doses may vary depending on the relative potency of individual antibodies, and in the case of combined use with other drugs, also on the relative potency of known drugs used to treat the disease.

[0146] In general, the doses range from 0.01 to 100 g per kg of body weight and can be administered once or more times a day, a week, a month or a year, and even once every 2-20 years. The dose and frequency of administration may vary depending on whether the agent is used prophylactically or therapeutically. In prophylactic applications, a relatively low dose is administered at relatively long intervals over a long period of time. Some patients receive the drug for the rest of their lives. In therapeutic applications, it is sometimes necessary to use a relatively high dose administered at relatively short intervals to slow down or slow down disease progression, although preferably until the symptoms of the disease are alleviated or subsided. At that time, the patient can be given the agent in a preventive scheme.

[0147] Although individual needs may vary, the determination of optimal ranges of effective amounts of pharmaceutical formulations is well within the skill of those skilled in the art. Dose heights for use in humans can be extrapolated from animal studies (Remington's Pharmaceutical Sciences, 20th ed., Gennaro, ed., Mack Publishing Co., Easton, Pennsylvania, 2000). In general, the dosage necessary to provide an effective amount of a particular pharmaceutical formulation that can be adjusted by a person skilled in the art will vary depending on the age, health status, physical capacity and weight of the patient, and the nature and severity of the disease or disorder, and also the frequency of obtaining a therapeutic agent,

[0148] The pharmaceutical compositions of the invention may be in pharmaceutical form using a pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable carriers include water, PBS, saline solutions (e.g. Ringer's solution), alcohols, oils, gelatins, as well as carbohydrates, e.g. lactose, amylose or starch, and further fatty acid esters, hydroxymethylcellulose and polyvinylpyrrolidine. The formulations may be sterilized and, if so desired, mixed with auxiliary agents, e.g. lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts affecting the osmotic pressure, buffers and colorants. Pharmaceutical carriers suitable for use in the present invention are known in the art (Remington's Pharmaceutical Sciences,

[0149] Incinerator pharmaceutical formulations that may conveniently be in the form of dosage units may be formulated according to conventional techniques well known in the pharmaceutical industry. These techniques include the step of combining the active ingredients with at least one pharmaceutical carrier. In general, these formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or particulate solid carriers or both. These formulations may be placed in single or multidose containers, e.g. in sealed ampoules and vials, and may be stored frozen or lyophilized, requiring only the addition of a sterile liquid carrier immediately prior to use.

[0150] Pharmaceutical compositions are generally formulated as sterile and essentially isotonic in accordance with all the principles of good manufacturing practice of the Food and Drug Administration.

[0151] Additional dosages of ίοΓηπ ^ φ, dosages and schedules of administration are available in the literature (Berkow et al., 1997, The Merck Manual of Medical Information, Home, ed., Merck Research Laboratories, Whitehouse Station, New Jersey; Goodman et al. , 1996, Goodman &amp; Gilman's The Pharmacological Basis of Therapeutics, 9th ed. McGraw-Hill Health Professions Division, New York; Ebadi, 1998, CRC Desk Reference of Clinical Pharmacology, CRC Press, Boca Raton, Florida; Katzung, 2001, Basic &amp; Clinical Pharmacology, 8th ed. Lange Medical Books / McGraw-HiH Medical Pub. Division, New York; Speight et al., 1997, Avery's Drug Treatment: A Guide to the Properties, Choice, Therapeutic Use and Economic Value of Drugs in Disease Management, 4th ed. Adis International, Auckland / Philadelphia, Pennsylvania).

[0152] The compositions of the present invention used as a pharmaceutical can be administered in monotherapy or together with other compounds or preparations used in the treatment of diseases in a malignant tumor, an infectious disease or an inflammatory disease. Examples of these compounds referred to herein as "additional compounds" or "additional preparations" are, inter alia, antibiotics, anti-cytokines, anti-asthmatics, anti-phospholipases (e.g. phospholipase inhibitors), vasodilators (e.g. adenosine, beta-adrenergic agonists, beta-adrenergic receptor antagonists, i.e. beta-blockers, alpha-blockers, diuretics, smooth muscle relaxants, nitrates, and angiotensin-converting enzyme inhibitors), biomolecules, cytostatics and chemotherapeutics. The pharmaceutical formulations of the invention may contain, for example, one or more additional compounds. In some embodiments, the antibody is conjugated to the additional compound.

[0153] Also disclosed herein are kits for, for example, treating a malignant tumor, an infectious disease or an inflammatory disease.

[0154] Antibody kits or antibodies of the invention and instructions for use of the kit in a method for treating a malignant tumor, an infectious disease or an inflammatory disease in a patient or a method for inhibiting the biological activity of a target antigen (e.g. GM-CSF). The kit may contain at least one additional compound. The kit may contain instructions and / or means for administering the antibody or antibody composition, e.g. by injection.

[0155] The entirety of the invention may be used to detect antigen in a biological sample, inter alia in blood serum. Any method known in the art, including flow cytometry, can be used. The biological sample may, for example, be incubated with the antibody of the invention, and the whole is then rinsed and incubated with the labeled secondary antibody. For example, the secondary antibody may be directed against the light chain and, for detection purposes, conjugated to FITC or phycoerythrin.

[0156] The antigen neutralizing activity exhibited by the antibodies of the invention may be tested using a biological antigen neutralization test or any other method known in the art. For example, neutralizing activity of antibodies can be assessed using antigen-dependent cell lines. Examples of GM-CSF-dependent cell lines are, inter alia, TF-1 and AML-193.

[0157] In the first biological assay for antigen neutralization, antigen-dependent cells are suspended in test medium, antigen or antigen pre-incubated for one hour with test antibodies or antibodies for isotype control. After the incubation period, growth inhibition is assessed using any method known in the art. For example, a Cell Titer reagent (Promega, Wisconsin, USA) may be added followed by further incubation followed by optical density measurement at 490 nm in a spectrophotometer and background medium subtraction from samples . The percentage of antigen neutralization is calculated from the following formula: 100 - (OD from Ig / OD without Ig) χ 100.

[0158] In another biological neutralization test, the antigen is mixed with the antibodies of the invention directed against the antigen. Antigen-dependent cells are added to the mixture and the whole is incubated. After the incubation period, the growth inhibition is measured. For example, the MTS deoxyribonucleic acid proliferation marker may be added, followed by measurement of the incorporation of the dye. Reduced incorporation of the dye in the presence of the antibody compared to the state in which the anti-antigen antibody is absent indicates the neutralization of the antigen.

[0159] According to another method, antigen-dependent cells are propagated in the presence of antigen, and then increasing amounts of antibody are added to the culture medium and evaluation of the neutralizing activity is performed as described above.

[0160] Antibodies of the invention and pharmaceutical formulations therefor may be used in methods for inhibiting the biological activity of a target antigen, e.g. GM-CSF, and for methods of treating a disease that may be, inter alia, malignant tumor, infectious disease or inflammatory disease, by administering pharmaceutical preparations with antibodies of the invention to a patient in need thereof. The GM-CSF biological activity is understood, inter alia, binding to the GM-CSF receptor. Antibodies of the invention and pharmaceutical formulations therefor may be used in methods used, e.g., as part of a prophylaxis or treatment of a disease that may be, inter alia, a malignant tumor, an infectious disease or an inflammatory disease.

[0161] Antibodies of the invention and pharmaceutical preparations therefor may be used in medical methods suitable for use in human and non-human animals. Non-human animals that benefit from the invention include domestic animals, exotic animals (e.g. zoological animals) and farm animals. Ideally, non-human animals in the above sense are mammals.

[0162] Antibodies of the invention may be useful in any acceptable pharmaceutical form, e.g. in the form of capsules, tablets, aqueous suspensions, solutions, etc. These antibodies may also be administered parenterally, i.e., by the following routes: subcutaneously, intravenously , intramuscularly, in ear, intraarticular, intrasystemic, intrasternal, intranasal, externally, intrathecally, externally, for lesion and intracranial - injection or infusion techniques. Generally, these antibodies will be administered as intramuscular or intravenous injections.

[0163] The inventors may provide an important or in combination with a pharmaceutically acceptable carrier to which adjuvants, vehicles and excipients may be acceptable.

[0164] The methods of the invention may be administered by, after or together with other therapeutics, For example, the antibodies of the invention may be administered as the sole ingredient or in combination with an additional compound.

[0165] The inventors may be administered as a homogeneous mixture of conjugated or non-conjugated antibodies or as a non-homogeneous mixture of conjugated and non-conjugated antibodies.

[0166] Eeczema efficacy can be resold in various ways. In an exemplary embodiment, the efficacy of the treatment of a malignant tumor, an infectious disease or an inflammatory disease is determined based on the finding of a slowing progression of the disease. In other embodiments, the measure of effectiveness of treatment is to improve the patient's condition, including symptoms such as weight gain, strength regain, reduced pain, normal development, and subjective patient relationships indicating improvement in health.

To facilitate the understanding of the present invention and its practice, several examples will be set forth below in which at least some disclosed aspects of the invention are described based on experimental work being carried out during its development. Any examples presented below that do not fall within the scope of the claims are provided for reference purposes only.

Example No. 1

Preparation of antigen-specific human monoclonal antibodies against GM-CSF

Materials and methods [0168] Human lymphocytesB, ex vivo immunization and culture

In all the procedures used, the cells were grown in a 5% CO2 atmosphere at 37 ° C. Leukopomas were obtained from healthy people vaccinated with tetanus toxoid (TT). PBMCs were purified using Ficoll-Plaque (Amersham BioSciences), CD19 + B lymphocytes and CD4 + T lymphocytes were isolated from PBMCs using the EasySep® kit for selecting human CD4 cells and human CD19 cells, respectively (StemCell Technologies), and then mixed to form a pool B lymphocytes / T lymphocytes (BT4 cells). BT4 cells were grown in full RPMI1640 medium (Invitrogen, California, USA), which contained 10% heat inactivated human AB serum (Nabi, Florida, USA), 2 mM L-glutamate,

0.1 mM non-essential amino acids, 1 mM sodium pyruvate, 55 μΜ 2-mercaptoethanol (Invitrogen, California, USA).

[0169] To perform ex vivo immunization, BT4 cells were co-cultured in the presence of T and B cell epitopes. Briefly, BT4 cells were seeded to a density of 10⁶/ ml in a full RPMI medium containing 1 Lf / ml tetanus toxoid (TT) (Cylex, Maryland, USA) in the presence of irradiated autologous PBMC at a 1: 1 ratio to obtain activated T lymphocytes (pool T). In order to obtain antigen-activated B lymphocytes (pool B), BT4 cells were plated to a density of 3 χ 106 cells / ml in a full RPMI medium containing 10% human AB serum, 5% conditioned medium from activated T lymphocytes, 20 U / ml IL-2 , 0.5 ng / ml IL-6, 100 U / ml IL-10 (PrepoTech, New Jersey, USA) and 250 ng / ml peptide cocktail (bio-World, Ohio, USA) representing different regions of the target antigen, synthesized in such a way that they contain both T and B epitopes as previously described in the literature (Zafiropoulos et al., The Journal o. "Immunological Methods 1997, 200: 181-90).

[0170] Selected B-cell epitopes for GM-CSF were the following sequences: EHVNAIQEARRLLNL (SEQ ID NO: 3), STQPWEHVNAIQEAR (SEQ ID NO: 4), MASHYKQHCPPTPET (SEQ ID NO: 5).

[0171] T and B pools were separately cultured for 7 days and then co-cultured (106 cells / ml) in a 1: 1 ratio on a single layer of irradiated CHO nutrient cells, in a full RPMI medium containing 10% heat inactivated human AB and 400 human serum. j./ml IL-4 (PrepoTech, New Jersey, USA). After five days, co-cultured T and B pools were fused to obtain hybridomas as described.

[0172] For the purposes of PAP-bearing immortal B -immhaimocytes, 100 ml of bw was processed in the PBMC purification cycle. Lymphocytes were cultured by 7 to 10 dtb in a medium RPMI medium containing 10% heat inactivated fetal bovine serum (FBS) (JHR Biosciences, Kansas, USA), 2 ng / ml IL-4 (PeproTech, New Jersey, USA), 2 mM L-glutamate, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, 55 μΜ 2-mercaproethanol (Invitrogen, California, USA), 50 pg / ml transferrin, 5 ng / ml phorbol acetate acetate (PMA, phorbol myristate acetate) and 0.5 pg / ml cyclosporin A (Sigma, Missouri, USA) in the presence of irradiated CHO nutrient cells. Subsequently, lymphocyte electrophoresis was performed as described below.

[0173] Cell fusion and ELISA screening of antigen-responsive hybridomas

Human B lymphocytes used to obtain monoclonal antibodies intended for human administration may be a potential carrier for viral transmission. Fusion partner cells and PBMCs from healthy donors were pre-screened by PCR to confirm the absence of viral DNA, including DNA belonging to Type 1 and Type 2 immune deficient viruses, hepatitis B and C viruses, cytomegalovirus, type 6 herpervirus and Epstein virus barr. The lymphocytes were fused with K6H6 / B5 cells (ATCC, Virginia, USA) using a CYTOPULSE CEEF-50 (Cyto Pulse Sciences, Inc., Maryland, USA) at a ratio of lymphocytes to K6H6 / B5 cells as 1: 1.

[0174] After fusion, cells were seeded into flat bottom 96-well microplates at a concentration of about 5,000 cells / well in complete RPMI medium containing 10% heat inactivated FBS, 2 mM L-glutamate, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, 55 μΜ of 2-mercaptoethanol (Invitrogen, California, USA), 100 μΜ of hypoxanthine, 0.4 μΜ of aminopterin and 16 μΜ of thymidine (HAT, Sigma, Missouri, USA). The medium was replaced every week, and the HAT selection was maintained until the screening for antigen reactivity (3-5 weeks).

[0175] To detect the reactivity of the monoclonal antibodies to the antigen, ELISA screenings were performed automatically using a BIOMEK FX liquid dispensing system integrated in the plate washer and spectrophotometer. Briefly, the microplates were coated at room temperature for 6 hours with on-site recombined antigen at 50 μΙ / well (1 μg / ml GM-CSF) diluted in coating buffer (50 mM bicarbonate, pH 9.4). The plates were then blocked with binding buffer [1% BSA (Sigma, Missouri, USA) and 0.05% Tween 20 (BioRad, California, USA) in PBS] for 2 hours at room temperature. The plates were washed once with wash buffer (0.05% Tween 20 in PBS) and the hybridoma supernatant at 50 μΙ / well was transferred to ELISA plates. The binding reaction was carried out at room temperature for 2 hours. The plates were then washed 4 times and 100 μl horseradish peroxidase (HRP) conjugated goat anti-human IgG + M (Jackson ImmunoResearch Laboratories, Pennsylvania, USA) diluted to 1: 10,000 in binding buffer was added and the reaction was performed in room temperature for 1.5 hours. In the last step, the plates were washed 4 times, and then SureBlue substrate (KPL, Maryland, USA) was added for 10 minutes at 100 μΙ / well. The reactions were stopped by adding 1 N sulfuric acid at 50 μΙ / well, followed by absorbance at 450 nm. horseradish peroxidase) (Jackson ImmunoResearch Laboratories, Pennsylvania, USA) diluted 1: 10,000 in binding buffer and the reaction was carried out at room temperature for 1.5 hours. In the last step, the plates were washed 4 times, and then SureBlue substrate (KPL, Maryland, USA) was added for 10 minutes at 100 μΙ / well. The reactions were stopped by adding 1 N sulfuric acid at 50 μΙ / well, followed by absorbance at 450 nm. horseradish peroxidase) (Jackson ImmunoResearch Laboratories, Pennsylvania, USA) diluted 1: 10,000 in binding buffer and the reaction was carried out at room temperature for 1.5 hours. In the last step, the plates were washed 4 times, and then SureBlue substrate (KPL, Maryland, USA) was added for 10 minutes at 100 μΙ / well. The reactions were stopped by adding 1 N sulfuric acid at 50 μΙ / well, followed by absorbance at 450 nm.

[0176] Analysis performed by fluorescence activated cell sorting (FACS)

The Ig binding and cell washing steps were performed using ice-cold binding buffer (DPBS without calcium and magnesium, 0.5% BSA), the reactions were set in Vdenne microplates, and the samples were analyzed using a FACSAria apparatus (BD Biosciences, New Jersey,

USA). For the purposes of the FACS experiment presented in Figure 3A 10⁶ mouse anti-GM-CSF hybridoma cells (Mull9 / 2) were placed with GM-CSF at 100 ng / reaction and incubated with human anti-GM-CSF E5 monoclonal antibody. Binding of monoclonal antibodies specific for GM-CSF was detected using 10 μg / ml FITC goat anti-human Ig (SouthernBiotech, Alabama, USA). For the FACS experiment shown in Figure 3B, hybridoma cells E10 were washed and plated at 500,000 cells / well in a volume of 90 μΐ. Then 10 μl of phycoerytin-labeled GM-CSF (R &amp; D System, Minnesota, USA) was added to each well and the cells were incubated on ice for one hour. As unmarked competition for GM-CSF, hybridoma cells were pre-incubated with recombinant human GM-CSF at 5 μg / ml (PeproTech, New Jersey, USA) at room temperature for one hour, three times rinsed, and then before being analyzed, they were incubated with PE-GM-CSF in the manner described above. For the needs of the FACS experiment presented in Figure 4, A431 and A431K5 cells (donated free of charge by Dr. Ira Pastan of the National Cancer Institute) were stained using either C12 monoclonal antibodies or normal human IgM (Jackson ImmunoResearch Laboratories, Pennsylvania, USA) at 10 μg / ml. diluted in binding buffer and reactions were carried out as described above.

[0177] Bioassay of GM-CSF neutralization

The GM-CSF-dependent cell line of human TF-1 erythroid leukemia (ATCC, Virginia,

USA) were grown in complete RPMI1640 medium (see above) containing recombinant human GM-CSF at 10 ng / ml (PeproTech, New Jersey, USA). On the day before the experiment TF-1 cells were expanded in 0.1% FBS in the absence of GM-CSF. The famed cells were rinsed twice, resuspended in test medium and plated into 96-well microplates at a concentration of 10,000 cells / well. The wells contained either a test medium, GM-CSF at 100 pg / ml, or GM-CSF pre-incubated for one hour with test antibodies or antibodies for isotype control at the concentrations indicated in the legends for figures. After 3 days, 40 μl of Cell Titer reagent (Promega, Wisconsin, USA) was added to each well and the plates were further incubated at 37 ° C for one hour. The optical density (OD) was measured at a wavelength of 490 nm in a spectrophotometer, subtracting the medium background for all samples. The percentage of GM-CSF neutralization was calculated from the following formula: 100 - (OD from Ig / OD without Ig χ 100).

[0178] Switchover of antibody classes

Hybridoma cells were washed once with 10 ml of PBS, resuspended in complete RPMI medium, plated into flat bottom 96 well microplates and incubated at 37 ° C in a 5% CO2 atmosphere. After four days, the cells were resuspended by pipetting and then 100 μΐ of the suspension was transferred to 20 ELISPOT plates (Millipore, Massachusetts, USA) coated with anti-human IgG (H + L) (Jackson ImmunoResearch, Pennsylvania, USA) at a concentration of 2.3 μg / ml. . The remaining cells in tissue plates were fed with an additional 100 μl of complete RPMI medium. After overnight incubation, the ELISPOT plates were washed three times with 0.05% Tween in PBS (PBST), and then 100 μl of goat anti-human IgG (H + L) conjugated to HRP at 2 μg / ml was added and the plates incubated for 1 hour at room temperature. shaking. Plates were washed three times with PBST, and then 100 μl of AEC substrate solution (Sigma, St. Louis, Missouri, USA) was added to the wells and incubated for 90 minutes at room temperature with shaking. The substrate was aspirated and the plates rinsed with distilled water and allowed to air dry. Clones from wells showing positive spots (indicative of IgG production) were expanded. The above step was repeated sequentially by plating positive clones at concentrations of 1000, 100, 10 and 0.25 cells / well while simultaneously monitoring positive wells until identification of the IgG-secreting monocell colony. and the plates were rinsed with distilled water and allowed to air dry. Clones from wells showing positive spots (indicative of IgG production) were expanded. The above step was repeated sequentially by plating positive clones at concentrations of 1000, 100, 10 and 0.25 cells / well while simultaneously monitoring positive wells until identification of the IgG-secreting monocell colony. and the plates were rinsed with distilled water and allowed to air dry. Clones from wells showing positive spots (indicative of IgG production) were expanded. The above step was repeated sequentially by plating positive clones at concentrations of 1000, 100, 10 and 0.25 cells / well while simultaneously monitoring positive wells until identification of the IgG-secreting monocell colony.

[0179] Fermentation using broad-porous membranes and a stirred tank bioreactor

Cells were plated at a concentration of 2.5 χ 10^five/ ml into a 2L bioreactor (B Braun Stat B-DU ) containing 11 serum free medium HyQCDM4NS0 (HyClone, Utah, USA), maintaining glucose concentration at 6 g / L and glutamine at 4 mM. Controlled setpoints were: pH 7.1, dO₂ 40% air saturation, temperature 37 ° C and shaking rate 80 rpm. 2 ml samples were taken daily: 1 ml for counting cells using the Cedex instrument and 1 ml for determination of Ig concentrations by ELISA. For the fermentation using wide-band membranes 10^eight viable cells were plated into a FiberCell system (Bellco, New Jersey, USA) containing 15 ml of complete RPMI medium and re-fed in an assembled tank containing 1 l of fresh medium when the cells consumed 50% glucose.

[0180] Inhibition of the repair of mismatched bases to increase the genetic diversity of the hybridoma line

Hybridoma cells were propagated in complete RPMI medium (negative control) or complete RPMI medium containing 250 μΜ or 500 μΜ of an anthracene compound inhibiting repaired mismatched rules (MMR). Cells were passaged at a 1: 5 dilution every three to four days in fresh medium with or without morphocene, and after three weeks, cells were harvested and resuspended at 2 χ 10⁶ in FACS buffer (1% BSA in PBS). Cells were stained with goat anti-human immunoglobulin FITC conjugated to Jackson ImmunoResearch at 10 pg / ml for 30 minutes on ice. The cells were rinsed with 10 ml of ice-cold FACS buffer and resuspended in 3 ml of FACS buffer. Then 10 μΐ of the Via-Probe solution (Becton Dickinson, Franklin Lakes, New Jersey, USA) was added for 5 minutes on ice, and viable cells were sorted for strong surface staining with immunoglobulin using the FACSAria cell sorter (Becton Dickinson). The goal was to sort the cells constituting 5% of the subpopulation showing the strongest surface staining with immunoglobulin. To select for the clones with elevated titers, FACS sorted cells were seeded into U-bottom 96-well plates and incubated for one week at 37 ° C in a 5% CO2 atmosphere. 50 μΐ supernatant was taken from the wells and analyzed for IgM production by ELISA using goat anti-human IgM + G plates. As part of the internal control, the contents of 3 wells of each ELISA plate were seeded with the addition of 50 μΐ human IgM (Jackson ImmunoResearch) at a concentration of 10 ng / ml. The OD values ​​obtained at 450 nm were normalized to the mean values ​​found in the internal control wells. Wells showing high IgM signals were expanded for further analysis. To analyze the microsatellite instability (MSI) microsatellite instability) DNA was extracted from parental cells or cells treated with morphocene using the Qiagen DNeasy Tissue kit (Qiagen). The poly (A) BAT (7) repeat marker was amplified using a fluorescently labeled BAT-26-F D4 primer (5'-tcaccatccattgcacagtt-3 ') (SEQ ID NO: 20) and BAT-26-R (5'ctgcgagaaggtactcaccc-3'). ) (SEQ ID NO: 21) and high purity polymerase pfuUltra ™ (Stratagene, California, USA), performing reactions under the following incubation conditions: 5 minutes at 95 ° C; 9 cycles of 1 minute at 94 ° C, 1 minute at 60 ° C and 2 minutes at 72 ° C at a temperature of 1 ° C hybridization at each cycle; 30 cycles of 1 minute at 94 ° C, 1 minute at 52 ° C and 2 minutes at 72 ° C; final extension for 10 minutes at 72 ° C. Single copies of the label allele were obtained using the DNA dilution leading to amplicon obtaining in only 50% of the PCR reaction. The PCR products were diluted 1: 10 with a CEQ sample loading solution and then placed on a Beckman chemistry analyzer CEQ8000 for fragment analysis.

[0181] Preparation of antigen-specific human monoclonal antibodies

Ex vivo immunizations were performed using cryopreserved B lymphocytes obtained from volunteers (healthy donors) as described above, or B lymphocytes were obtained from human subjects whose sera were characterized by a high titre of monoclonal antibodies exhibiting specificity for the antigen of interest. The rationale for the second of the above-mentioned methods is the possibility that a portion of antigen-specific monoclonal antibodies could be a product of a pathological immune response (as, for example, in patients with autoimmune diseases) or in vivo immune responses to tumor, microbial or vaccine antigens.

[0182] Antigenically reactive human monoclonal antibody clones were identified following fusion of ex vivo immunized cells from hybridoma libraries obtained from B lymphocytes from patients suffering from PAP. Four hybridoma lines - E5 (IgM), G7 (IgM), E1O (IgG) and G9 (IgG) - were selected for further study, and the human monoclonal antibodies produced by them were tested for specificity by ELISA. Figure 1 shows that human monoclonal antibodies E5, G7 and E10 only reacted with human GM-CSF and did not react with any of the other ten unrelated test antigens, including the mouse GM-CSF, which is 53% identical to the human homolog . Similar results were obtained with the G9 hybridoma. In order to confirm the specificity of the above-mentioned human monoclonal antibodies, FACS analyzes were performed. Human GM-CSF was allowed to bind to the surface of the mouse hybridoma cells on which the membrane monoclonal antibodies expressing specificity for human GM-CSF on another epitope are expressed. The monoclonal antibodies E5 bound to the surface of these cells, under the conditions described, as indicated by the shift in fluorescence intensity (Figure 2A, bottom panel). The results obtained demonstrate the ability of E5 monoclonal antibodies to bind native human GM-CSF. In the absence associated with GM-CSF cells, the E5 monoclonal antibodies did not cross-react with any of the cell membrane proteins expressed in these hybridoma cells (Figure 2A, middle panel). Similarly, monoclonal antibodies E10 showed high specificity in the FACS analysis. Also, since the association of monoclonal antibodies E10 with the cell membrane of hybridoma cells was found, FACS analysis showed the ability of these antibodies to bind soluble GM-CSF labeled with phycoerythrin (PE) (Figure 2B, middle panel). Binding specificity was demonstrated by pre-incubating E10 hybridoma cells with an excess of unlabelled GM-CSF (Figure 2B, lower panel).

[0183] Switching de novo class of human monoclonal antibodies

Using the two strategies described above, human monoclonal IgG and IgM antibodies were directed against a variety of human and non-human antigens. Although the majority of therapeutic antibodies available on the market belong to the IgG isotype, oncological studies evaluating the use of potentially therapeutic IgM monoclonal antibodies have shown tumor regression in vivo (16, 17). The clinical responses obtained in these studies can be attributed to the ability of IgM to bind strongly and activate the complement pathway and effectively kill tumor cells. IgG binds to Fc receptors on macrophages and NK cells and can therefore mediate ADCC-type reactions directed against cancer cells. Both IgG, and IgMs with identical specificity (the same antigen and epitope) can be tested for the best in vivo pharmacological activity. In cases where the isotype of the IgG isotype was the preferred isotype, a fast and efficient procedure was used to switch the de novo IgM class (see "Materials and Methods"). Using line E5 as an example, a cell subpopulation was identified in which the class was switched to IgG isotype under the applied culture conditions. In the case of IgG produced by E5 line cells, an identical nucleotide sequence was shown in the variable region and a similar reactivity to GM-CSF (Figure 3) as for IgM produced by the parental E5 lineage cells. a quick and efficient procedure was used to switch the IgM de novo class (see "Materials and Methods"). Using line E5 as an example, a cell subpopulation was identified in which the class was switched to IgG isotype under the applied culture conditions. In the case of IgG produced by E5 line cells, an identical nucleotide sequence was shown in the variable region and a similar reactivity to GM-CSF (Figure 3) as for IgM produced by the parental E5 lineage cells. a quick and efficient procedure was used to switch the IgM de novo class (see "Materials and Methods"). Using line E5 as an example, a cell subpopulation was identified in which the class was switched to IgG isotype under the applied culture conditions. In the case of IgG produced by E5 line cells, an identical nucleotide sequence was shown in the variable region and a similar reactivity to GM-CSF (Figure 3) as for IgM produced by the parental E5 lineage cells.

[0184] The biological activity of human anti-angiotic and non-human subjects

The pharmacological properties sought in therapeutic monoclonal antibodies targeting soluble mediators of diseases include the ability to neutralize growth factors. As already mentioned, one example of these factors is GM-CSF, which is one of the mediators of RA (9-11). The ability of human monoclonal antibodies to block the action of GM-CSF was evaluated. This was done using a cellular bioassay in which the growth of human erythroid cell leukemia (TF1) cells depends on the presence of the above-mentioned cytokine in the culture medium. As shown in Figure 4, both E10 and G9 lead to statistically significant inhibition of GM-CSF-dependent cell growth, while human IgGs that control isotype did not induce this effect. The difference in potency between E10 and G9 correlates well with the apparent affinities of these antibodies of 870 pM and 14 pM respectively. E5 monoclonal antibodies showed only minimal neutralizing activity corresponding to lower affinity (5 nM).

[0185] Evaluation of the titres and stability of hybridomas secreting human monoclonal antibodies

One of the important properties of the monoclonal antibody production line is the stability of immunoglobulin secretion during the entire batch cycle. According to one scenario assuming a cycle length of about two months, a line doubling every 24 hours would go through about 60 generations from thawing to collecting cells. The E5 line was used as a model to test the monoclonal antibody titers and the stability of the hybridoma production using our method. The 3D2 clone obtained from this line showed a doubling time of 24 hours and was again cloned by limiting dilution after more than two months of uninterrupted culture. The frequency of clone production was determined by ELISA, measuring immunoglobulin concentrations in their conditioned media normalized to cell density. Figure 5A shows that all tested E5-3D2 subclones secrete immunoglobulins in high concentrations, which indicates homogeneous retention of immunoglobulin generation in this cell population after 60 generations. The generation of immunoglobulins was then evaluated at a small scale (15 ml) using a broad-band membrane system. The cells were inoculated in a cassette with a wide-band membrane and fed continuously in an assembled reservoir containing one liter of fresh medium. Starting from the 5th day, all conditioned medium from the cassette (15 ml) was harvested once a day and replaced with fresh medium. Fermentation was carried out for an additional 4 days, whereas once a day immunoglobulin titers were determined by ELISA using an immunoglobulin standard of known concentration. In the described period of 4 days, a cumulative titer of 1.2 g / l was recorded. Between 8 and 9. the consumption of glucose reached its maximum (2 g / l / day), indicating that cells tolerate extremely high concentrations of cells well. Production efficiency on a 1-liter scale was also evaluated using a batch fed-mode method using a bioreactor with a mixer. The cells from the frozen ampoule were first thawed, inoculated in a shake flask, and then plated in a bioreactor with a stirrer (Bauer) containing 1 liter of serum-free medium. Fermentation was performed to a decrease in cell viability below 60% (6 days). The amount of immunoglobulin produced and cell density was recorded between the 1st and 5th day, and the results obtained are given in Figure 5B. During the logarithmic phase of growth (from the first to the 4th day), specific productivity was found at 24 pg / cell / day with a doubling time of 23.4 hours on average,

[0186] Genetic optimization of the hybridoma secreting human monoclonal antibodies by regulating the repair of mismatched bases

The usefulness of improving the quality of cell lines that produce monoclonal antibodies using a process called morphogenesis process that relies on transient MMR regulation has been demonstrated in the literature (Nicolaides et al., Genomics 1995, 30: 195-206; Nicolaides et al., Molecular and Cellular Biology 1998, 18: 1635-41.). After increasing the genetic diversity of the cell pool using this method, high pass throughputs were performed to identify subclones with higher titers, affinities (Grasso et al., BioProcess International 2004, 2: 58-64; Nicolaides et al., The Annals of the New York Academy of Sciences 2005,1059: 111) or a higher rate of multiplication (L. Grasso - personal observation). E5 cells were subjected to a morphogenetic process to demonstrate the ability to enhance phenotypic diversity in cell lines secreting monoclonal antibodies obtained using our hybridoma targeting strategy. MMR inhibition was monitored by detecting microsatellite instability (MSI) in the poly (A) BAT repeat marker. In 3 out of 24 BAT alleles analyzed in cells exposed to morphogenesis, changes were found including deletions and insertions of single nucleotides (deletions illustrated in Figure 6A). MSI was not detected in any of the 24 BAT alleles in the parental cells. Then, the parental cells and the cells subjected to the morphogenetic process were sown with the method of limited dilutions into microplates. Cells were allowed to secrete monoclonal antibodies for one week and their immunoglobulins by ELISA were determined in their conditioned medium. The frequency of clones with an optical density greater than 1 (high level of immunoglobulin secretion) was calculated for the total number of screened clones (3763 in the parental pool and 2437 in the morphogened pool), indicating an increase in the mentioned frequency by 260% (p = 0.0014) in population subjected to the morphogenic process (Figure 6B).

Summary [0187] This study provides a feasible strategy for the development of human monoclonal antibodies for immunotherapy using an optimized ex vivo immunization process and immortalizing human B cells combined with inhibition of repair of mismatched bases. Thanks to this method, highly specific and biologically active monoclonal antibodies secreted by stable hybridoma lines can be obtained.

[0188] According to the methods of the invention, stable production of monoclonal antibodies was obtained over more than 60 doubling periods and production of more than 1 gram of monoclonal antibodies per liter during a 4-day fermentation cycle in broad-band membranes, indicating that the hybridoma cells obtained by the methods give for use in flow systems and potentially in large scale production. Furthermore, the hybridomas obtained in this process showed good parameters in fed-batch fermentation, suggesting the potential usefulness of these cell lines in commercial applications. In conclusion, the platform process presented here offers an alternative approach to the rapid and cost-effective development of fully human high-quality antibodies for use in immunotherapy.

Example No. 2

Preparation of fully human anti-GM-CSF antibodies; PAP cell isolation [0189] In adult patients with P 2&gt; PP phthalytic poxemia, phospholipids and surfactant proteins accumulate in the alveoli.

It has been hypothesized that the development of PAP occurs due to the inability of alveolar macrophages and type II epithelial cells to remove excess surfactant. As described above, the function of GM-CSF in controlling the homeostasis of pulmonary surfactant has been established in a mouse experimental model and, by analogy, in humans it is a determinant of pathology. In addition, anti-GM-CSF neutralizing antibodies were found in PAP patients, which implies that this cytokine is the causative agent of this disease. It is not known whether the autoimmune response described above is specific to GM-CSF or not. It has been shown, however, that in a subgroup of patients with PAP, the state of health is improved as a result of the use of GM-CSF. This fact is an argument supporting the hypothesis,

[0190] Isolation of antibodies specific for GM-CSF

Peripheral blood mononuclear cells (PBMCs) were isolated from PAP patients. Briefly, B-lymphocytes from PAP patients were obtained from whole blood. Whole blood was diluted with the same volume of PBS - / -, and the whole was gently mixed by inversion of the container. 25 ml of diluted blood was layered onto a 50 ml tube containing 25 ml of Ficoll-Paque medium (Amersham Biosciences AB, Uppsala, Sweden). The tubes were centrifuged at 2000 rpm for 30 minutes at room temperature. PBMCs were collected from the solution boundary layer using a 10 ml pipette, transferred to new 50 ml tubes and washed twice with PBS - / - solution. The PBMC pellet was resuspended in 10 ml of ACK cytolytic buffer (150 mM NH4G, 10 mM KHCO3, 0.1 mM Na2EDTA, pH 7.2),

[0191] Fusion of B lymphocytes from PAP patients with myeloma cell line for hybridoma

Enriched B lymphocytes from PAP patients were fused with various myeloma cells (human-mouse heterofibridoma, ATCC, Virginia, USA), CBF-7 (human-mouse heterozygybody) cells, HEK 293, human myeloma cells using the method described below. B-lymphocytes and fusion partner cells should have a good viability (&gt; 90% of cells should be viable and be in the logarithmic growth phase). Cells of both types were counted in their original media and mixed in 1 ml tubes at a 1: 1 ratio, followed by centrifugation at 1000 rpm for 6 minutes at 4 ° C. The cells were washed three times with 10-15 ml of cold CPFM medium (CYTOPULSE fusion medium, Cyto Pulse Sciences, Maryland, USA). The final pellet was resuspended at a concentration of 10 × 10⁶ cells / ml of CPFM medium.

Electrofusion was performed using a CYTOPULSE CEEF-50 (Cyto Pulse Sciences, Inc., Maryland, USA). The fusion parameters were optimized according to the manufacturer's guidelines and empirical guidelines, resulting in a fusion yield of one hybrid per 5,000 cells undergoing pulsation. After fusion, cells were seeded into flat bottom 96-well microplates at a concentration of about 5,000 cells / well in complete RPMI medium containing 10% heat inactivated FBS, 100 μΜ hypoxanthine, 0.4 μΜ aminopterin and 16 μΜ thymidine (HAT, Sigma, Missouri, USA). . The medium was replaced every week, and selection using HAT was continued until antigen reactivity ceased.

[0192]

Screening by ELISA

In order to detect the reactivity of monoclonal antibodies to antigen, ELISA screening was carried out automatically using the BIOMEK FX liquid dispensing system integrated in the plate washer and spectrophotometer. Briefly, the microplates were coated at room temperature for 6 hours with on-site recombinant antigen at 50 μΙ / well (1 gg / ml GM-CSF) diluted in coating buffer (50 mM bicarbonate, pH 9.4). The plates were then blocked with binding buffer [3% BSA (Sigma, Missouri, USA) and 0.05% Tween 20 (BioRad, California, USA) in PBS] for 2 hours at room temperature. The plates were washed once with wash buffer (0.05% Tween 20 in PBS) and 50 μΙ / well of the hybridoma supernatant was transferred to ELISA plates. The binding reaction was carried out at room temperature for 2 hours. The plates were then rinsed 4 times and 100 μΐ of goat anti-human IgG + M HRP-conjugated (Jackson ImmunoResearch Laboratories, Pennsylvania, USA) diluted 1: 10,000 in binding buffer was added and the reaction was carried out at room temperature for 1.5 hours. In the last step, the plates were washed 4 times, and then SUREBLUE substrate (KPL, Maryland, USA) was added for 10 minutes at 100 μΐ / well. The reactions were stopped by adding 1 N sulfuric acid at 50 μΙ / well, followed by absorbance at 450 nm. 10,000 in binding buffer and the reaction was carried out at room temperature for 1.5 hours. In the last step, the plates were washed 4 times, and then SUREBLUE substrate (KPL, Maryland, USA) was added for 10 minutes at 100 μΐ / well. The reactions were stopped by adding 1 N sulfuric acid at 50 μΙ / well, followed by absorbance at 450 nm. 10,000 in binding buffer and the reaction was carried out at room temperature for 1.5 hours. In the last step, the plates were washed 4 times, and then SUREBLUE substrate (KPL, Maryland, USA) was added for 10 minutes at 100 μΐ / well. The reactions were stopped by adding 1 N sulfuric acid at 50 μΙ / well, followed by absorbance at 450 nm.

[0193] FACS analysis and sorting

In each study, immunoglobulin binding and cell wash steps were performed using ice-cold binding buffer (DPBS without calcium and magnesium, 0.5% BSA). PBMCs or hybridoma cells were washed and plated at 500,000 cells / well. Anti-human CD3, CD19 and CD20 labeled with FITC and phycoerythrin (PE) were then added (SouthemBiotech, Alabama, USA) and phycoerythrin-labeled GM (CS) (R &amp; D System, Minnesota, USA) (diluted 10 to 100 times) and incubated on ice for an hour. The cells were washed three times with binding buffer and then analyzed or sorted using a FACSAria apparatus (BD Biosciences, New Jersey, USA). The results of the isotype analysis of monoclonal antibodies E10 are shown in Figure 9.

[0194] Bioassay of GM-CSF neutralization

The human-GM-CSF dependent human TF1 erythroblast leukemia cell line (ATCC, Virginia, USA) was amplified in complete RPMI1640 medium (see above) containing recombinant human GM-CSF at 10 ng / ml (PeproTech, New Jersey, USA). On the day before the experiment, TF-1 cells were placed in 0.5% FBS in the absence of GMCSF. The starved TF-1 cells were harvested and rinsed twice with test medium (simple RPMI medium with 0.5% BSA). Cells were resuspended in assay medium and plated into 96-well microplates at a concentration of 10,000 cells / well. The wells contained either a test medium, GM-CSF at 100 pg / ml, or GM-CSF pre-incubated for one hour with test antibodies or antibodies for isotype control at the concentrations indicated in the figure. After 3 days, 40 μΐ of Cell Titer reagent was added to each well (Promega, Wisconsin, USA) and plates were further incubated at 37 ° C for one hour. The optical density (OD) was measured at 490 nm in a spectrophotometer, subtracting the medium background from all samples. The percentage of GM-CSF neutralization was calculated from the following formula: 100 - (OD from Ig / OD without Ig) χ 100. Figure 4 shows the GM-CSF-dependent inhibition of TF-1 cell line growth by fully human monoclonal antibodies.

[0195] Inhibition of the repair of mismatched bases to increase the genetic diversity of cell lines created by hybridomas

Inhibiting the repair of mismatched rules can lead to genetically diverse sister cells characterized by increased antibody production, faster growth or greater activity of the produced antibodies. To increase the activity of the produced monoclonal antibodies and to accelerate cell growth, hybridoma cells were propagated in complete RPMI medium (negative control) or full RPMI medium containing 250 μΜ or 500 μΜ of the mismatched repair inhibitor treated with morphocene, i.e. 9,10-dimethylanthracene ( MP Biomedicals, California, USA). Cells were passaged at a 1: 5 dilution every three to four days in fresh medium with or without morphocene, and after three weeks, cells were harvested and resuspended at 2 χ 10⁶ cells / ml in FACS buffer (1% BSA in PBS). Cells were stained with goat anti-human immunoglobulin FITC conjugated to Jackson ImmunoResearch at 10 pg / ml for 30 minutes on ice. The cells were rinsed with 10 ml of ice-cold FACS buffer and resuspended in 3 ml of FACS buffer. Then 10 μl of the Via-Probe solution (Becton Dickinson, Franklin Lakes, New Jersey, USA) was added for 5 minutes on ice, and viable cells were sorted for strong surface staining with immunoglobulin using a FACSAria cell sorter (Becton Dickinson). The goal was to sort the cells constituting 5% of the subpopulation showing the strongest surface staining with immunoglobulin. This population was exposed to a week-long expansion in the presence or absence of a chemical mismatch repair inhibitor (MMR) and this procedure was repeated two additional times. In order to select for clones with elevated titers, FACS sorted cells were seeded into U-bottom 96-well plates at a concentration of 0.8 cells / well in 200 μΐ of complete RPMI medium. The plates were then incubated for one week at 37 ° C in a 5% CO2 atmosphere. 50 μl of the supernatant was removed from the wells and analyzed for IgM production by ELISA using goat anti-human IgM + G plates. As an internal control, the contents of 3 wells of each ELISA plate were seeded with the addition of 50 μl human IgM (Jackson In order to select for clones with elevated titers, FACS sorted cells were seeded into U-bottom 96-well plates at a concentration of 0.8 cells / well in 200 μΐ of complete RPMI medium. The plates were then incubated for one week at 37 ° C in a 5% CO2 atmosphere. 50 μl of the supernatant was removed from the wells and analyzed for IgM production by ELISA using goat anti-human IgM + G plates. As an internal control, the contents of 3 wells of each ELISA plate were seeded with the addition of 50 μl human IgM (Jackson In order to select for clones with elevated titers, FACS sorted cells were seeded into U-bottom 96-well plates at a concentration of 0.8 cells / well in 200 μΐ of complete RPMI medium. The plates were then incubated for one week at 37 ° C in a 5% CO2 atmosphere. 50 μl of the supernatant was removed from the wells and analyzed for IgM production by ELISA using goat anti-human IgM + G plates. As an internal control, the contents of 3 wells of each ELISA plate were seeded with the addition of 50 μl human IgM (Jackson using tiles covered with goat anti-human IgM + G for this purpose. As an internal control, the contents of 3 wells of each ELISA plate were seeded with the addition of 50 μl human IgM (Jackson using tiles covered with goat anti-human IgM + G for this purpose. As an internal control, the contents of 3 wells of each ELISA plate were seeded with the addition of 50 μl human IgM (Jackson

ImmunoResearch) at a concentration of 10 ng / ml. The OD values ​​obtained at 450 nm were normalized to the mean values ​​found in the internal control wells. Wells showing high IgM signals were expanded for further analysis. For the analysis of MSI, DNA was extracted from parental cells or cells treated with an MMR inhibitor using a Qiagen DNeasy Tissue kit (Qiagen). The poly (A) BAT repeat marker was amplified using a fluorescently labeled primer mBAT-26-F D4 (5'-tcaccatccattgcacagtt-3 ') (SEQ ID NO: 20) and mBAT-26-R (5'-ctgcgagaaggtactcaccc-3') ( SEQ ID NO: 21) and high fidelity polymerase pfuUltra ™ (Stratagene, California, USA), performing reactions under the following incubation conditions: 5 minutes at 95 ° C; 9 cycles of 1 minute at 94 ° C, 1 minute at 60 ° C and 2 minutes at 72 ° C at annealing temperature of 1 ° C every cycle; 30 cycles of 1 minute at 94 ° C, 1 minute at 52 ° C and 2 minutes at 72 ° C; final extension for 10 minutes at 72 ° C. Single copies of the label allele were obtained using the DNA dilution leading to amplicon obtaining in only 50% of the PCR reaction. The PCR products were diluted 1: 10 with a CEQ sample loading solution and then placed on a Beckman chemistry analyzer CEQ8000 for fragment analysis. (Blake et al. "Stepwise deletions of polyA sequences in mismatch repair-deficient colorectal cancers." The American Journal of Pathology 2001, 158: 1867-70.) 30 cycles of 1 minute at 94 ° C, 1 minute at 52 ° C and 2 minutes at 72 ° C; final extension for 10 minutes at 72 ° C. Single copies of the label allele were obtained using the DNA dilution leading to amplicon obtaining in only 50% of the PCR reaction. The PCR products were diluted 1: 10 with a CEQ sample loading solution and then placed on a Beckman chemistry analyzer CEQ8000 for fragment analysis. (Blake et al. "Stepwise deletions of polyA sequences in mismatch repair-deficient colorectal cancers." The American Journal of Pathology 2001, 158: 1867-70.) 30 cycles of 1 minute at 94 ° C, 1 minute at 52 ° C and 2 minutes at 72 ° C; final extension for 10 minutes at 72 ° C. Single copies of the label allele were obtained using the DNA dilution leading to amplicon obtaining in only 50% of the PCR reaction. The PCR products were diluted 1: 10 with a CEQ sample loading solution and then placed on a Beckman chemistry analyzer CEQ8000 for fragment analysis. (Blake et al. "Stepwise deletions of polyA sequences in mismatch repair-deficient colorectal cancers." The American Journal of Pathology 2001, 158: 1867-70.) Single copies of the label allele were obtained using the DNA dilution leading to amplicon obtaining in only 50% of the PCR reaction. The PCR products were diluted 1: 10 with a CEQ sample loading solution and then placed on a Beckman chemistry analyzer CEQ8000 for fragment analysis. (Blake et al. "Stepwise deletions of polyA sequences in mismatch repair-deficient colorectal cancers." The American Journal of Pathology 2001, 158: 1867-70.) Single copies of the label allele were obtained using the DNA dilution leading to amplicon obtaining in only 50% of the PCR reaction. The PCR products were diluted 1: 10 with a CEQ sample loading solution and then placed on a Beckman chemistry analyzer CEQ8000 for fragment analysis. (Blake et al. "Stepwise deletions of polyA sequences in mismatch repair-deficient colorectal cancers." The American Journal of Pathology 2001, 158: 1867-70.) "Stepwise deletions of polyA sequences in mismatch repair-deficient colorectal cancers". The American Journal of Pathology 2001, 158: 1867-70.) "Stepwise deletions of polyA sequences in mismatch repair-deficient colorectal cancers". The American Journal of Pathology 2001, 158: 1867-70.)

Example No. 3

Preparation of E10 monoclonal antibody showing high specificity for GM-CSF. Several antibodies were obtained from B -immigocytes derived from patients with PAP. These antibodies belonged to IgM and IgG isotypes. For each of these antibodies, specific binding to GM-CSF was found using the biological specificity assays described herein. Human anti-GM-CSF monoclonal antibody IgG1 E10 was obtained from B cells derived from PAP patient, which lymphocytes had previously been fused with myeloma cells, and then, using the ELISA method as described above, were screened for monoclonal antibodies directed against human GM-CSF.

[0197] A total of 49.5 million PBMCs were isolated from blood collected from one patient on PAP (91 ml of whole blood). The viability of these cells was 99.0%. The results of the FACS analysis are shown in Table 2. B cells were expanded to culture about 25 million PBMCs in complete RPMI1640 medium (10 ml) with IL-4 at 2 ng / ml (PeproTech), transferrin at 50 pg / ml ( Sigma), PMA at a concentration of 5 ng / ml (Sigma) and cyclosporin A at a concentration of 0.5 pg / ml (Sigma) with nutrient cells. After 11 days of culture, 12 million cells remained, which were then electrophobied with myeloma cells (using the CytoPulse CEEF-50). The FACS analysis results are shown in Table 2.

Table 2. Preparation of monoclonal antibody E10 and analysis by FACS

<td></td><td>FITC-CD3(%)</td><td>FITC-CD20 (%)</td><td>PE-GM-CSF (%)</td><td>CD20 / GM-CSF</td>

<td>PBMC</td><td>62.6</td><td>14.4</td><td>5.9</td><td>6.3</td>

<td>11. day hodll</td><td>for example,</td><td>17.0</td><td>07</td><td>01</td>

[0198] Fused cells were grown in RPMI1640 medium (Invitrogen, California,

USA) containing 10% heat inactivated FBS (JRH Biosciences, Kansas, USA), Ligutamine at 200 mM (Invitrogen, California, USA), non essential amino acids at a concentration of 10 mM (Invitrogen, California, USA), sodium pyruvate at 100 mM (Invitrogen, California, USA), Pen-Strep (Invitrogen, California, USA), 2-mercaptoethanol at 55 mM (Invitrogen, California, USA) and 1xHAT (Sigma, Missouri, USA).

[0199] After 12 days of culture, approximately 48% of the hybridoma cultures showed an increase. The hybridomas were then screened using a GM-CSF-specific ELISA assay with recombinant GM-CSF (PeproTech, New Jersey, USA). Several clones were isolated, including GM-CSF-positive clones. These clones were re-tested by ELISA to confirm that they show specificity for recombinant GM-CSF and not for tetanus toxin (TT). Clone 4E10 showed a specific reaction with GM-CSF, but not with TT. The 4E10 clone was then subcloned. After 3 weeks, the cultured subclones were screened by ELISA to confirm persistence of GM-CSF specificity and then characterized as described below.

[0200]

Characterization of E10 antibodies

In order to determine the specificity of anti-GM-CSF E10 antibodies, an antigen-specific ELISA was performed using a series of antigens (Figure 8). In Figure 8, binding to the following antigens was compared: hIL-1α, hIL-2, hIL-3, hIL-4, hIL-5, IL-6, hIL-13, hGM-CSF, mGM-CSF, BSA and TT. In subsequent studies, the binding of anti-GM-CSF antibodies to the following ligands was compared: human GM-CSF, human mesothelin, SEB, BGG, CAB, HEL, TT, BSA, goat IgG, human mucin and mouse IgG (data not shown). In both studies, 4E10 antibodies reacted exclusively with human GM-CSF.

[0201] To standardize the isotype of E10 antibodies, a standard analysis was performed using anti-human IgG, IgG1, IgG2, IgG3, IgM, Lk and LA Fc antibodies. Analysis showed that the antibody secreted by clone 4E10 is the IgG1 and kappa antibody (see Figure 9).

[0202] The ability of E10 antibodies to neutralize GM-CSF under in vitro conditions was tested using the TF-1 cell line, the survival and growth of which depends on this cytokine (see Figure 8). The human-GM-CSF dependent human TF-1 erythroid cell line (ATCC, Virginia, USA) was propagated in complete RPMI1640 medium containing recombinant human GM-CSF at 10 ng / ml (PeproTech, New Jersey, USA). On the day before the experiment, TF-1 cells were placed in 0.1% FBS in the absence of GM-CSF. The starved TF-1 cells were harvested and rinsed twice with test medium (simple RPMI medium with 0.5% BSA). The cells were resuspended in assay medium and plated into 96-well microplates at a concentration of 10,000 cells / well. The wells contained either test medium, GM-CSF in an amount of 100 pg / ml, or GM-CSF pre-incubated for one hour with test antibodies or antibodies for isotype control at concentrations ranging from 20 pg / ml to 0.315 pg / ml. After 3 days, 40 μl of Cell Titer reagent (Promega, Wisconsin, USA) was added to each well and the plates were further incubated at 37 ° C for one hour. The optical density (OD) was measured at 490 nm in a spectrophotometer, subtracting the medium background from all samples. The percentage of GM-CSF neutralization was calculated from the following formula: 100 - (OD from Ig / OD without Ig) χ 100. The 4E10 antibody demonstrated the ability to neutralize GM-CSF under in viiro at a concentration of 100 pg / ml. USA) and the plates were further incubated at 37 ° C for one hour. The optical density (OD) was measured at 490 nm in a spectrophotometer, subtracting the medium background from all samples. The percentage of GM-CSF neutralization was calculated from the following formula: 100 - (OD from Ig / OD without Ig) χ 100. The 4E10 antibody demonstrated the ability to neutralize GM-CSF under in viiro at a concentration of 100 pg / ml. USA) and the plates were further incubated at 37 ° C for one hour. The optical density (OD) was measured at 490 nm in a spectrophotometer, subtracting the medium background from all samples. The percentage of GM-CSF neutralization was calculated from the following formula: 100 - (OD from Ig / OD without Ig) χ 100. The 4E10 antibody demonstrated the ability to neutralize GM-CSF under in viiro at a concentration of 100 pg / ml.

[0203] E10 antibodies were subjected to western blot analysis to determine if they cross-react with human recombinant GM-CSF. As seen in Figure 10, the E10 antibodies cross-react with human GM-CSF under reducing conditions.

[0204] The binding affinity of E10 antibodies was determined using a BIACORE analyzer as described below. A binding constant for antibodies of E10 of about 870 pM was obtained (Figure 11).

Example No. 4

Preparation of G9 monoclonal antibody showing high specificity for GM-CSF [0205] Anti-GM-CSF monoclonal antibody of IgG1 G9 genus was obtained from B-cells derived from PAP disease, which lymphocytes had previously been fused with K6 or CBF-7 cells, and then using the ELISA method as described above, screened for monoclonal antibodies directed against human GM-CSF.

[0206] A total of 49.5 million PBMCs were isolated from blood collected from one patient on PAP (91 ml of whole blood). The viability of these cells was 99.0% (data not shown). B lymphocytes were expanded by culturing approximately 25 million PBMCs in complete RPMI1640 medium (10 ml) with the addition of IL-4 at a concentration of 2 ng / ml (PeproTech), transferrin at 50 gg / ml (Sigma), PMA at a concentration of 5 ng / ml (Sigma) and cyclosporin A at a concentration of 0.5 gg / ml (Sigma) with nutrient cells. After 11 days of culture, 12 million cells remained, which were then electrophoresed with myeloma cells (using the CytoPulse CEEF-50).

[0207] Fused cells were grown in RPMI1640 medium (Invitrogen, California,

USA) containing 10% heat inactivated FBS (JRH Biosciences, Kansas, USA), Ligutamine at 200 mM (Invitrogen, California, USA), non essential amino acids at a concentration of 10 mM (Invitrogen, California, USA), sodium pyruvate at 100 mM (Invitrogen, California, USA), Pen-Strep (Invitrogen, California, USA), 2-mercaptoethanol at 55 mM (Invitrogen, California, USA) and 1xHAT (Sigma, Missouri, USA).

[0208] After 12 days of culture, approximately 48% of hybridoma cultures showed an increase. The hybridomas were then screened using a GM-CSF-specific ELISA assay with recombinant GM-CSF (PeproTech, New Jersey, USA). Several clones were isolated, including GM-CSF-positive clones. These clones were re-tested by ELISA to confirm that they show specificity for recombinant GM-CSF and not for tetanus toxin (TT). The G9 population showed a highly specific reactivity with GM-CSF, but not with TT. Subsequently, the G9 population was subcloned to obtain a pure culture. After 3 weeks, the cultured subclones were screened by ELISA to confirm persistence of GM-CSF specificity and then characterized as described below.

[0209]

Characterization of G9 antibodies

In order to determine the specificity of anti-GM-CSF G9 antibodies, an antigen-specific ELISA was performed using a series of antigens (Figure 8). In Figure 8, binding to the following antigens was compared: hIL-1α, hIL-2, hIL-3, hIL-4, hIL-5, IL-6, hIL-13, hGM-CSF, mGM-CSF, BSA and TT. In subsequent studies, the binding of anti-GM-CSF antibodies to the following ligands was compared: human GM-CSF, human mesothelin, SEB, BGG, CAB, HEL, TT, BSA, goat IgG, human mucin and mouse IgG (data not shown). In both studies, G9 antibodies reacted exclusively with human GM-CSF.

[0210] Standardized analysis using anti-human IgG, IgG1, IgG2, IgG3, IgM, Lk and LA Fc antibodies was performed to determine the design of the G9 antibody. Analysis showed that the G9 antibody is the IgG1 and kappa antibody (Figure 12).

[0211] Bioassay of neutralization by antibody G9

The ability of G9 antibodies to neutralize GM-CSF in vitro was tested using the TF-1 cell line, the survival and growth of which depends on this cytokine (Figure 4). The human-GM-CSF dependent human TF-1 erythroid cell line (ATCC, Virginia, USA) was propagated in complete RPMI1640 medium containing recombinant human GM-CSF at 10 ng / ml (PeproTech, New Jersey, USA). On the day before the experiment TF-1 cells were expanded in 0.1% FBS in the absence of GM-CSF. The starved TF-1 cells were harvested and rinsed twice with test medium (simple RPMI medium with 0.5% BSA). The cells were resuspended in assay medium and plated into 96-well microplates at a concentration of 10,000 cells / well. The wells contained either test medium, GM-CSF in an amount of 100 pg / ml, or GM-CSF pre-incubated for one hour with test antibodies or antibodies for isotype control at concentrations ranging from 20 gg / ml to 0.315 pg / ml. After 3 days, 40 μl of Cell Titer reagent (Promega, Wisconsin, USA) was added to each well and the plates were further incubated at 37 ° C for one hour. The optical density (OD) was measured at 490 nm in a spectrophotometer, subtracting the medium background from all samples. The percentage of GM-CSF neutralization was calculated from the following formula: 100 - (OD from Ig / OD without Ig) χ 100. The G9 antibody demonstrated the ability to neutralize GM-CSF activity in vitro at a concentration of 100 pg / ml. USA) and the plates were further incubated at 37 ° C for one hour. The optical density (OD) was measured at 490 nm in a spectrophotometer, subtracting the medium background from all samples. The percentage of GM-CSF neutralization was calculated from the following formula: 100 - (OD from Ig / OD without Ig) χ 100. The G9 antibody demonstrated the ability to neutralize GM-CSF activity in vitro at a concentration of 100 pg / ml. USA) and the plates were further incubated at 37 ° C for one hour. The optical density (OD) was measured at 490 nm in a spectrophotometer, subtracting the medium background from all samples. The percentage of GM-CSF neutralization was calculated from the following formula: 100 - (OD from Ig / OD without Ig) χ 100. The G9 antibody demonstrated the ability to neutralize GM-CSF activity in vitro at a concentration of 100 pg / ml.

[0212] G9 antibodies were subjected to western blot analysis to determine if they cross-react with human recombinant GM-CSF. As seen in Figure 10, G9 antibodies cross-react with human GM-CSF under reducing conditions.

[0213] The affinities of G9 antibodies were determined using a BIACORE analyzer as described above. A binding constant was obtained for G9 antibodies of around 11-17 pM (Figure 13).

Example No. 5

Nucleotide sequences encoding fully human anti-GM-CSF antibodies G9 and E10 [0214] Antibody G9

Nucleotide and amino acid sequences for the fully human anti-GM-CSF G9 antibody were obtained by standard methods. Briefly, all RNA was isolated from the G9 hybridoma using the Trizol reagent (Invitrogen) according to the manufacturer's instructions. The template cDNA was synthesized using the Superscript II reverse transcriptase (Invitrogen) according to the manufacturer's instructions. To amplify the light and heavy chain variable regions, PCR reactions were performed using Herculase DNA polymerase (Stratagene), using primers # 22 and # 23 for the light chain and primers # 24 and # 25 for the heavy chain. The PCR products were cloned into the pCR4-TOPO vector (Invitrogen), transformed into E. coli Mach1 cells, and transformants were selected on LB plates with kanamycin. Colonies were screened for inserts using the same primer pairs as above, and four positive colonies were used to obtain template DNA for DNA sequence determination using TempliPhi reagent (GE Healthcare). The DNA inserts were sequenced using primers # 26 and # 27 using a Beckman Coulter DTCS sequencing reagent followed by data acquisition and analysis on a Beckman Coulter CEQ 2000 analyzer. To add the leader peptide sequence to the light chain, the positive clone was amplified again with primers No. 28 and No. 23 using Herculase DNA polymerase. In order to obtain a full-length heavy chain (sequence No. 11), including the leader peptide sequence, PCR was performed using primers # 29 and # 30 using the original cDNA as template. The obtained PCR product was subjected to TA cloning, transformed into Mach1 cells and - as described above - positive clones were identified. Full-length heavy chain cDNA for G9 antibody was sequenced using primers No. 25, No. 26, No. 31, No. 32, No. 33, No. 34, No. 27 and No. 30 using template DNA obtained using TempliPhi reagent. The resulting DNA sequences for the full-length heavy chain component of the G9 antibody (SEQ ID NO: 11) and for the full-length light chain component of the G9 antibody (SEQ ID NO: 15) are shown below. The anticipated translation products of sequences Nos. 11 and 15 are depicted as sequences, respectively, No. 9 and No. 13. The predicted translation products of sequences Nos. 10 and 14 are shown as sequences No. 8 and No. 12, respectively. The underscore fragments of sequence No. 11 and No. 15 represent the leader sequence added in PCR. Polynucleotide sequences No. 10 and No. 14 code for the heavy chain and light chain of the G9 antibody, respectively, without added leader sequences. The fragmented sequences 9 and 13 in small letters represent human leader peptides added in the PCR reaction. The underlined fragments of sequences No. 8, No. 9, No. 12 and No. 13 represent the CDR regions. The remaining fragments of sequences No. 8, No. 9, No. 12 and No. 13 represent the foundations of variable regions and constant regions. The heavy chain constant region begins with the WGQG amino acid sequence (amino acid at position 111 of sequence 8 or at position 130 of sequence 9),

[0215] Antibody E10

Nucleotide and amino acid sequences for fully human anti-GM-CSF E10 antibody were obtained by standard methods. Briefly, all RNA was isolated from the E10 hybridoma using the Trizol reagent (Invitrogen) according to the manufacturer's instructions. The template cDNA was synthesized using the Superscript II reverse transcriptase (Invitrogen) according to the manufacturer's instructions. To amplify light and heavy chain variable regions, PCR reactions were performed using Herculase DNA polymerase (Stratagene), using primers # 22 and # 23 for the light chain and primer # 24 and # 25 for the heavy chain. The PCR products were cloned into the pCR4-TOPO vector (Invitrogen), transformed into E. eoli Mach1 cells, and transformants were selected on LB plates with kanamycin. Colonies were screened for inserts using the same primer pairs as above, and four positive colonies were used to obtain template DNA for DNA sequence determination using TempliPhi (GE Healthcare). The DNA inserts were sequenced using primers # 26 and # 27 using a Beckman Coulter DTCS sequencing reagent followed by data acquisition and analysis on a Beckman Coulter CEQ 2000 analyzer. The obtained DNA sequences encoding the heavy chain variable region constituting the antibody E10 (SEQ ID NO: 17) and the full-length light chain constituting the antibody E10 (SEQ ID NO: 19) are shown below. The predicted products of the translation of sequences 17 and 19 are shown as sequences 16 and 18, respectively. The underlined fragments of sequences 16 and 18 represent CDRs. The remaining fragments of sequences No. 16 and No. 18 represent the foundations of variable regions and constant regions. The heavy chain constant region starts with the WGQG amino acid sequence (amino acid at position 115 of sequence No. 16), while the light chain constant region - on the amino acid sequence of FGQG (amino acid at position 98 of sequence No. 18).

Example No. 6

Epitope mapping for the anti-GM-CSF antibody [0216] For the mppowaniaepitopu GM-CSF with which the G9 antibody was incubated, a series of overlapping peptides including the full length of human GM-CSF were obtained (Figure 14). Briefly, 25 overlapping seven amino acids of the 12-mer peptides were designed including the human GM-CSF sequence (Gen. GenBank AAA52578, residues 14-144). The peptides were obtained as individual spots of 3.7 mm χ 3.7 mm by solid phase synthesis by attaching the carboxyl terminus of each peptide to the surface of the membrane of derivatized cellulose (SPOTs technology from Sigma-Genosys). A standard western blot analysis was performed to determine which peptides cross-react with the G6 antibody (Figure 15).₃) overnight at 4 ° C. A fresh blocking solution containing purified 10G9 antibody at a concentration of 1 mg / ml was added, and the paper was incubated overnight at 4 ° C. The paper was rinsed three times for 5 minutes in TBS-T (1xTBS, 0.1% Tween-20), and then incubated for 1 hour at a dilution of 1: 10,000 goat anti-human IgG (H + L) conjugated to HRP (Jackson ImmunoResearch, Cat. 109-035-088) in a diluent (5% BSA, 1xTBS, 0.1% Tween-20). The paper was developed using the SuperSignal West Femto ECL Substrate Kit (Pierce, Cat. No. 34095), followed by one-second exposure to BioMAX (Kodak). As a result of this method, peptides numbered 6, 13, 14 and 15 and probably peptide No. 23 were specifically recognized by the G9 antibody (corresponding sequences are: No. 18, No. 19, No. 20, No. 21 and No. 47).

[0217] The present invention is not limited to the embodiments described and schematised above, but may be subject to changes and modifications within the scope of the appended claims.

Sequence list

Seq. No. 1:

MMQ SL The LLG TVACSI SAPARSg 0 PSI QPWE HA IQE ARFLLLH The SRDTAAEMKE TVEV! SEMF DLQEF TC LQTRLE LYKGGLRGSL TKLKGFL ridASRYKQHCFF TPETSCATIA^itfE SFKEKLK □ FLL VIFfTDCPEFVQE

Seq. No. 2:

AFARSPSPSTQPWEHVNATQEAFRLLiNL3RD1AAEMH'ETVEVISEKFDLGEFTCLQ^rfRLELYKQ GLRGS LT KLKG? LTMMAS H YKGHCPPTFET SCATQ11TFESFKENLKDFLLVIPFDCWSPVQE

<td>Seq.</td><td>No.</td><td>3:</td><td>EHVNAIQEARRLLNL</td>

<td>Seq.</td><td>No.</td><td>four:</td><td>STQPWEHVNAIQEAR</td>

<td>Seq.</td><td>No.</td><td>five:</td><td>MASHYKQHCPPTPET</td>

<td>Seq.</td><td>No.</td><td>6:</td><td>LSTAVKELVENSLDAGATNIDLKLKDYGVDLIEVSDNGCGVEEENFE</td>

<td>Seq.</td><td>No.</td><td>7:</td><td>LRQVLSNLLDNAIKYTPEGGEITVSLERDGDHLEITVEDNGPGIPEEDLE</td>

Seq. 8: The predicted amino acid sequence in the heavy chain G9 EVQLVE £ GGGLVQPGG SLRL3 CAA SGFTF SRH.WM HWLRQVPGKGjćVHffVSR't Kai.GT SITYADSV CEMR "FI SRDNAN N TLFLCKN 3LFADDT ALYFCARAN SV WL RG ^ LF DYP and ug TFVTVS SAS T KGPS vFFLPTSSKSTSGGTAlPLGCLVKdYFPEPVTvBWNISGALi PSS TSGyHEFPAWiJSSGLYSLSSWTy SL (3 Τΐαν.Μ HKPSNTKV DKKVEP KSCDZKTΗ T CFFCFAPELLGGPSVFLFPPKFK DTL LI SPTPEVT CVWDVS HE DFE VKFRD "EVDGVE VHNAKTKFKEEQ EDS T ERD SVL TVL HQ DDULG KE EKC KVS NKALFAP IΕΚΤI SK.AKGQFRE P QV ETLPPSKDE L TKNQV SLT CLVKGFEP S DI AVE WS SD ' GQPENL EKT TPP VL DSD GEF FLE SKL TVDK S KDQOGNMF Ξ C SVMH E AL HNH E TGKSL S LS PGK *<a name="caption1"></a>Seq. No. 9: predicted amino acid sequence in the heavy chain of G9 (with the leader) mi # s; s ItSEh Q ^ \ ^ QE VC Ξ G GGI ^ i ^ t ^ PGG LL ^: L JlASCP ": TFSRWM ^^WLR0^vp^ GKGP

VD VS KINGAGT SIT YAD SVKGKFT and S REN ANNTLF L QLN SLRAD DT ALEFC akan SyWKGLFD ΪDGGGETVVTΞΞASSITK ¥^JS1VIPL &amp; FSXK5TS'GGTAALGILVKDYFPEIPET V5WNSGALTSGVHT F PAVLQ SS GLYS N 3 S DTVP SSSLG TQTYICNVNHKPSNTKVDKKVEPXS DKTH C TCPPCPAPE LLG GP SV? LFPP KPK DTLLIS K 'PE VT &lt;VVVDVSHEDPEVK NNWY V DGVES V HNAK KKF lEHEO TN 3 HERDV 3VLTL ™ L HQDWLNGKE || K &lt;Ckf S SDALLAA P EKT IS KA Q PRE Q QVE TLPPS RDELTKN QVSLTcLV ^ KGF ¢ IPSDIA \\\ »5NGQPEKHYKTT 'PPVLD5DG5EFLLSKLKT □ K5XWgbGHVGGI S VLIHJAL HNHE T QX SLSLS PGK *

Seq. 10: nucleic acid sequence in the chain cagżtgcagctggtggastctgggggaggcttagttcagccgggggggtccctgagactctcct GTGCAGCCTCTGGATTCACTTTCaGTAGACACTGGATGCaCTGGCTTCGCCAGGTTCCAGTTAA GGGGC CGGTCTGGGTC T Gtat CiULTłS CAC TGC TC TGGGACT CATAACC TAC GCGGACTCCGTG AGGGGCCGATTCACCATCTCCAGAGACAACGCCAACAACACACTGTTTCTGCAAATGAACAGTC

TGAGAGCCGACGACACGGCTCTTTATTTCTGTGCAAGAGCGAACAGCGTCTGGTTCCGGGGCCT

CTTT GAC TACTG GGGC CAGG GAAC C CC CG TCAC CGT CT CCT CAGCC TC CACCAAGGGC C CATCG gtcttccccctggcaccctcctcgaagaccacctgtgggggcacagcggccctgggctgcctgg T CAAG GAC TAC on the TCC CC GAACCGGTGACGGTGT CGT GGAAC T CAGGCGCCC ΤGACCAGCGGCGT GCACACCT TCCC GGCT GT CCTACAGT CC Γ CAGGAC TC TAC TC CCT CAGCAG CG TGGTGACCGTG CCC TCCAGCAGC TTGGGCAC CCAG CC TAGAT C TGCAACGTGAAT CACAAGCCCAGCAACAC CA AGGTGGACAAGAAAGTTGAGCCCAAATCITGTGACAAAACTCACACATGCCCACCGTGCCCAGC AC C TGAAC TCCT GGGG GGAC CG TCAG Γ TC TCC TT TC CC CCCCAAAACCCAAGGACACCCTCATG ATCTCCCGGACCCCTGAGGTCACATGCSrGGTGGTGGACGTGAGCCACGAAGACCCTGAgGTCA AGT TCAAC TGGTACGTGGAG GGCGTG GAGGTGCATAAT GCCA GACAAAGCC GCGG GAGGAGCA GTACAaIAGIACGTACCGTGTGGTCAGCGTCCTCAIIGTCITGCACCAGGACTGGITGAATGGC aaggagtacaag TGCAAGGT TCCA C C C AAACC CC TCC AGCC cccatcgagaaaac C ATCTC CA

AAJ iCCAAAGGGCAGC ^ ^ G CCGAGAACCACAG TGTAC ACC C TGCC CCCATCC CGGGATGAGC T GAC CAAGjAACCAGGTCCAGCTGACCTGCCTGGTCiAAAGGCTTCTArCCCAGCGACATCSeCGTGGAG TGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGiGCTGGACTCCGACG GTTTTϊ · TτTTTTTTCTCGCCCGTrTCTTGGGGCTCCGCGGCAGG^oneGGGCAGCAGGGG.CCCG^.TTrr τrccrGcrccGrGcrGτcrGCGGérτrτrGcccccτcccrccccGCGccGCGC &lt;^ ircrτcτrGrcr TTTGGCACTGC

Seq. 11: nucleic acid sequence in the heavy chain G9 (leader) aagcttgccgccącca •.-ggatggagcτGtatCatCCtcttcttGgtagc &amp; agagc-aCaG3tg 2acagagC.gziggtgcagctggtggagcctgggggaggcttagttcagccgggggggtccctGag actctcctgtgcagcctctggattcactttcagtagacactggatgcactggcttcgccaggtt CCAGGTAAGGGGCCGGTCTGGGTCTCACGTATCAATGGTGCTGGGACTTCCATAACCTACGCGG CTrTCGTGCGGGGTTGCTr CCGAGCCCCCG TTCCTCCTCTCT T TG TT C TGCCCCT

GCATA.GTTTGAGCGCCGAC G ICCiCC GGiCT C TTTCTTTCT G TGC A AGCGTGCA ^ TΆGCGTC^.TGGT^{C. whereas,}rT

TGGβGCCrTTTTGCTTCTrGGGGCTCGGGCCTTTTGGTTACTGTTTTTTCCGCTrTTC.TTCAGG GCC CATCGGTCTTCC CCCTGGCaCCCTCCtccaagagc acctctGGGGGCACAGCGGCCCTGGG CTGCCTGGTCAAGGACTACΤTCCCCGAACCGGTGACGGTGTCGTGGAACΓCAGGCGCCCTGACC AGCGG CCTGC AC ACCT TCCC G GCTGT CCTCCAS TC C TTCGG^.ATT CT ^ CTCC TrTCGTCGTGT GG TGACCGCGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCCGCAACGTGAATCACAAGCCCAG TCCTACTACGGT G GCCAAGJCCAGrTGAGCOCAA ATC TT GTGCTCCCCCTTCTCTCT GCCC CCCG TGCC TCGTCTTTGCAT T CCT GGGGGGACC T CCG TCT TCC TC TT CCCCC TCAAŁTTCACGGCTA

CCCTCACGATCTCCCGGACCCCTGAGGTCACATGCGTGGtGGTGGACGTGAGCCACGAAGACCC rGCGGrTCAGrrTCCTr GGT ACGTGGAC GGCGT SGAGGrGTAr * AATGCC AAGCTCACGTTGTGG GAGGCGCCGrCCCATCG TCCCG G CCC TGT CCG GGT CGT CC TCCC C GTCC TGCCC CAGGACrGGT TGCCT GGCCC-GGCG TATCAG TGTAAGGrT TTGAA.TAAAGTTTr C CCCCC CCCCArTGAGAAAAC CATCTCCAAAGCCAaAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCGGGGAT GCGCrGATTCCG_JCACCACK ^ TCAGCCTGACCôί »TCTG {α TCAAjCS ^ TREATTTTA ^ TGATCrTG -GTC GCGTGGGCGAGC jCC ·? GGACCCCGACC AC GC CC CCGT GCTGGC TC

CTCC GCCGGCT CC T TC T TCC rcrCTCG TCAGTr TCTTGrGGAT AAGAGCAGGrGGCCGCC.GGGG CCCG TCT TC TCCT GCT C CGT GAΪGTATGAGGT TCT GCACCŁCC CC TCTATG CCGCCGCGCTrTr -Ή TCT C CC GG: GAAATGIA ^ aATET

Seq. No. 12: the predicted amino acid sequence in the G9 light chain

EIVL TQ SFVTL 3 V SPGECV T LSTRASQSV ^ tlNLC¾y QQKL GQ GFSL T. 7 YGASTESCrD AND PAST S _GSGSETEFTLTI5SLQSEDFAVYYCQCrDKraFDTFGQG :: KLEIKRTVJUA&gt; SVFIF? P5DEQLKSG

TASWCLlNUFFFPREAK VQ^rK &lt;VDN? LQQ S GN S OE SVTECDSKDS TYS LESTLTL SKADYEKHKVY ACEVTHQGLSSPVTKSFWRGEC *

Seq. No. 13: predicted amino acid sequence in the G9 light chain (with the leader) mgwaciilrlvS.tatgvhsEIVLTQSPV rLSVSPGERVTlSCRftSQSV5iHlAWYY! QKLC; GGr^.R LU YGASTRATA DIPARTS GS GS E TEFT LTIS3LQ 5EDFAVYYCQ &lt;iYDKWFDTFGQG TKL EIKRT VAAFSVFiFPPSDEOLKSGTA3WCLLNNFYFREAKVQWKVDNALQSGNSQE3VTEQDSKDSTY SLSS1L T LSKADYEKKKVYACEVTHQG1SSFVTKSFNRGEC *

Seq. No. 14: nucleic acid sequence in the G9 light chain

GAAAT GC TGT TC TGACTCAG TCCAG TCAC CCTG TCTGTGTC TCCAGGGGAAAGAGTCACTCTCT CCTGCAGGGCCAGTCAGAGTGTTAGCACCAACT TAG CCTGG TATCAGCAGAAAC TTGGCCAGGG CCCAGGC T T T GCTCAT ΓATGGTGCATCCACCAGGGC GA GAT GT ATCC SC CA CAG AG GGTCAGTGGC TGGG TC T Γ GAGACAGAGT CACTC TCAC CAGCAG CCT CAT TCT GCAG GAAGAT T TTGCAGTTT A.TTACTGTCAGCAA.TArGAIAAGTGGCCGGA .CACTTTTCGCCAGGGGACCAA.GCTGGAGATCAA ACGAACTGTGGCTGCACCArCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAAT CTGGA AC TGCCTCTGTTG TGTGC CTGCTGAATAAC TTGTATCC CAGAGAGGCCAAAGTACAGT GAAGG G T CC T GGATAACGC GCAAT C TAAG GGG TCC G CAGGAGAGT TCACAGAGCAGGACAGCAAGGACAG CA CCTACAGC TGAGCAGGACCC TGACGC T C GAG GAAAGGAGAC TACCACAAACACAAAC TCTAC □ CC TCCGAACTCACCCAT CAGGGCCT GAGTTCGC CCGrCACAĄACACCTTCAACACCCCACACT GTTA

Seq. No. 1S: nucleic acid sequence in the G9 light chain (with the leader) aagct - g and cg. and ac c AT GG GAT G GAGCT G TATCAT CCT CTTCr TGGTAGCAAGA CCTAi; ACCTC TACACACCCAAATTCTCCTCACTCACTCTCCACTCACCC TCTCTCTCTCT CCACCCCAAACAC T CAC TCTCTCCTCCACCCCCACTCAGAGTCTTAGCACCAACΓTAGCCTGGTArCAGCAGAAAOTT C GCCAGGGT CC CAGGCTCCTCAT TTATGGTGCATC CAC CA ^ GCCAC TGATAT CCCACCCACCT T GAG TGGCAGT GGG TC ^ AGAC ^ GA ^ T CAC TCTC ACCAT GAGCAGCCTCCACTCTGAAGATT TTG CAGT T TAT TAC TG TCACCAATATCATAAC TGGC CGGACACT TTT ^ CCA-GG GGAC CAAGC TGG AGAT CAAAC GAAC TGTGG Cr G CAC CATCT G TCT T CATC TTCC CGCCAT C ZGA ^ AaCA ^! ' T GA AATCTGGAAC TGCC TC TGT TGT G TGC C TGC T GAATAACTTCTA TCC CACAGAGCCCAAAGTACA □ TCCAACCTCCAΓAACCCCCWCCAATC □ GGTAACTCCCACCACACTCTCACACAGCA □ GACAGC AACCACAcCAC CTACAGCCTCAGCAGC ACCCTGACGC TC AGC AAAGCAGAC TACGAGAA AC ACA

AAGICTACGCCTGCGAAGICACCCATCAGOGCCTGAGTTCGCCCGUCAcAAAGAGCITCAACAG

GGGAGAG TGT TAAgaattc

Seq. No. 16: predicted amino acid sequence in the E10 heavy chain

QVQLE GGGWQFGRSLRL E 3 S * CAASGFTFTM GtlHWV RQaPGK CL EWLALISYDGHRQYYAPSV K £ £ RFTVSRPNPNNTLHLEMK L. &amp; AEDs IYYCARGAxGVLLWFGPLSWFDPWGQGILVTVSSASI KGPS VFP L SK APS STS GGTAALGC LVKPYF PSFVTVSWN S GAL TS GVI1T FPAVLQSSGLYSLSS WTVF SSSLGTQT YLCWVNH KPSNT XVd

Seq. No. 17: nucleic acid sequence in the E10 heavy chain

CAGG TAC AGCTGGAGGAG Γ CAGGGGGAGGCGT GGT CCAGCC T GGGAG GTCCCT CAGAC TCT CCT GT GCAGC GTCTG GATT CACC TI CAC ΤΛΑΤ TATG GCATGCACTGG GICCCC CAG GC TCCAGGCAA

GGGACTCGAGTGGCTGGCACTCAIATCCTATGATG GAAAIAGGCAAIACTATGCAGACTCCGTG AAGGGCC GAT TC ACCGT -C CAGAGAC AATCCCAAGAACACAC GCATC T Γ G GAGATGAAGAGCC ICCGAGCCGAAGAC TCGGC TATATAT TACTG TGCCAGAGGCGC IGGGGTΑΓ TACT GT GG TC T GG C CGACT TAIC TGGT TCGACC CTGGGGCCAGGGAACCC C T GGT CAC and CCAC CGTCTCCTCAGCC C

AAGGGCCCATCGGTCIT CC CC CCCGGCAC CC TCCT C CAAGCACC TCTGGGG GCACAGC GGCC C 'IGGG CTGCC TGGT CAAGGAC TAC CCCCGAACCG G TGACGGTGT CGTGGAACICAGGC GCC CT GACCAGCGGCGT G CACACCTTCCCGGCTG CACACCTTCCCGGCTG TCCT ACAGT GCTC AGGACT C TAC TCC CT CAGCAGC

GT GG TGAC CGTGC CCT C CAG CGCT TGGG CACCCAGACC TRAY TC TGCAAC GTGAAT CACAAG C

CCAGCAACACCAAGGTGGAC

Seq. No. 18: predicted amino acid sequence in the E10 light chain

DIQMTQ SPSNLS A SVGP RVTII CRASQM1NTWLAWYQHK PGKPPKLRIYQASILESCTP SRJ SG SGSGTIFTITISSLOPEDFGTYYCQQHMSYFYT^F G GGTKLEINRTVAAP SVFIFPPS DEOLKSG TA S WCL LNNFYP REAKVQWKVPNALQ S GWSQE SVTEQP S MDSlYSLSSUTL SKADYE KHKVY ACEVTHQGI33PVTKSFNRGEC

Seq. No. 19: nucleic acid sequence in the E10 light chain

GATATCC AGATGACCCAG TC TCC TT CCAACCIGIC T GCAT C TG TAGGAGACAGAGTC ACAATCA CTIG TCG G GCCAG TCAAAATAITAATAC C TGGCTG GCCT GGIATCAGCACAAACCAGGGAAACC C CC TAAGC TCCGGATATATCAGGCGTCTACGTTAGAAAG TGGG GT CCC TTCAAGGIT CAGCGGC agt Gg rt c tgggacgatat t cactc tc accatcag c AGC ctgcagcct grade gatttt ggaac it ATIAC TGC CAACAGAATAAIAGT TACCCGTACACT TT TGG CCAGGGGACCAAG C TGGAGAICAA C CGAACT G TGGG T GCAC CAT C TGTCT CTATCTICC CGCC ATCT GAT GAGCAGT TGAAATCIGGA

ACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGG T GG ATA ACGCCC TCCAATCG- AACT GGT CCC AGG AG AG CACAGAGCAGGACAGCA TGT TGG AC AG CACCTACAGCCT CAGCAGC ACCCTGACGC TGAG CAAAGCAGACT ACG AGAAAC ACAAAGT CTAC GCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGASAGT GTTA

Seq. No. 20: TCACCATCGATTG CAC AGTT

Seq. No. 21: CTGCGAGAAGGTACTCACCC

Seq. No. 22: GAHRTYSWGHTGACBCAGTCTCC

Seq. No. 23:

GATCGAATTCTTAACACTCTCCCCTGTTGAAGCTCTTTGTGACGGGCGAGCTCAGGCC

Seq. No. 24: AGGTRCAGCTGBWGSAGTCEiG

Seq. No. 25: GTCCACCTTGGTGTTGCTGGGCTT

Seq. No. 26: AGCGGATAACAATTTCACACAGG

Seq. No. 27: CGCCAGGGTTTTCCCAGTCACGAC

Seq. No. 28:

GATCGGATCCGCCGCCACCATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACA ggtgtacacagcgaaattgtgctgactcagtctcc

Seq. No. 29: GATCGAATTCTCATTTCCCGGGAGACAGGGAGAGG

Seq. No. 30:

GATCGGATCCAAGCTTGCCGCCACCATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACA gctacaggtgtacacagcgaggtgcagctggtggagtctgg

Seq. No. 31: GGACAAGAAAGTTGAGCCCA

Seq. No. 32: TGCAAGGTCTCCAACAAAGC

<td>Seq.</td><td>No.</td><td>33:</td><td colspan="2">CCTGGTTCTTGGTCAGCTCA</td>

<td>Seq.</td><td>No.</td><td>34:</td><td colspan="2">GGCACGGTGGGCATGTGTGA</td>

<td>Seq.</td><td>No.</td><td>35:</td><td>ARRLLNLSRDTA</td><td></td>

<td>Seq.</td><td>No.</td><td>36:</td><td>TRLELYKOGLRG</td><td></td>

<td>Seq.</td><td>No.</td><td>37:</td><td>YKOGLRGSLTKL</td><td></td>

<td>Seq.</td><td>No.</td><td>38:</td><td>RGSLTKLKGPLT</td><td></td>

<td>Seq.</td><td>No.</td><td>39:</td><td>KENLKDFLLVIP</td><td></td>

<td>Seq.</td><td>No.</td><td>40:</td><td>(G9 H CDR1)</td><td>GFTFSRHWMH</td>

<td>Seq.</td><td>No.</td><td>41:</td><td>(G9 H CDR2)</td><td>RINGAGTSITYADSVRG</td>

<td>Seq.</td><td>No.</td><td>42:</td><td>(G9 H CDR3)</td><td>ANSVWFRGLFDY</td>

<td>Seq.</td><td>No.</td><td>43:</td><td>(G9 L CDR1)</td><td>RASQSVSTNLA</td>

<td>Seq.</td><td>No.</td><td>44:</td><td>(G9 L CDR2)</td><td>GASTRAT</td>

<td>Seq.</td><td>No.</td><td>43:</td><td>(G9 L CDR3)</td><td>QQYDKWPDT</td>

<td>Seq.</td><td>No.</td><td>46:</td><td>(E10 H CDR1)</td><td>GFTFTNYGMH</td>

<td>Seq.</td><td>No.</td><td>47:</td><td>(E10 H CDR2)</td><td>LISYDGNRQYYADSVKG</td>

<td>Seq.</td><td>No.</td><td>48:</td><td>&lt;E10 H CDR3}</td><td>GAGVLLWFGDLSWFDP</td>

<td>Seq.</td><td>No.</td><td>49:</td><td>(E10 L CDR1)</td><td>RASQNINTWLA</td>

<td>Seq.</td><td>No.</td><td>50:</td><td>(E10 L CDR2)</td><td>GASTLES</td>

<td>Seq.</td><td>No.</td><td>51:</td><td>(E10 L CDR3)</td><td>QQNNSYPYT</td>

<td>Seq.</td><td>No.</td><td>52:</td><td>(G9 H FWRl)</td><td>EVQLVESGGGLVQPGGSLRLSCAAS</td>

<td>Seq.</td><td>No.</td><td>53:</td><td>(G9 H FWR2J</td><td>WLRQVPGKGPVWVS</td>

<td>Seq.</td><td>No.</td><td>54:</td><td>(G9 H FWR3)</td><td>RFTISRDNANNTLFLQMNSLRADDTALYFCAR</td>

<td>Seq.</td><td>No.</td><td>55:</td><td>(G9 L FWRl)</td><td>ΕIVLTCS PVTLSVS PGERVTLSC</td>

<td>Seq.</td><td>No.</td><td>56:</td><td>(G9 L FWR2)</td><td>WRONGLESS</td>

<td>Seq.</td><td>No.</td><td>57:</td><td>(G9 L FWR3)</td><td>DIPARFSGSGSETEFTLTISSLQSEDFAVYYC</td>

<td>Seq.</td><td>No.</td><td>58:</td><td>(E10 H FWRl)</td><td>QVQLEESGGGWQPGRSLRLSCAAS</td>

<td>Seq.</td><td>No.</td><td>59:</td><td>(E10 H FWR2)</td><td>WVRQAPGKGLEWLA</td>

<td>Seq.</td><td>No.</td><td>60:</td><td>(E1C H FWR3)</td><td>RFTVSRDNPNNTI.HLEMKSLRAEDSAIYYCAR</td>

<td>Seq.</td><td>No.</td><td>61:</td><td>(E10 L FWRl)</td><td>DIQMTQSPSNLSASVGDRVTITC</td>

<td>Seq.</td><td>No.</td><td>62:</td><td>{E10 L FWR2)</td><td>WYQHKPGKPPKLRIY</td>

<td>Seq.</td><td>No.</td><td>63:</td><td>(E10 L FWR3)</td><td>GVPSRPSGSGSGTIFTLTISSLQPDDFGTYYC</td>

Sequence list &lt;110&gt; Sass, Phillip M. Nicolaides, Nicholas C. Grasso, Luigi Li, Jian Chao, Qimin Routhier, Eric Ebel, Wolfgang &lt;120&gt; ANTIGEN GM-CSF PEPTIDES AND ANTIBODY AGAINST GM-CSF &lt;130&gt; MOR-0688 &lt;150&gt; US 60 / 774,500 &lt;151&gt; 2006-02-17 &lt;150&gt; US 60 / 771,251 &lt;151&gt; 2006-02-08 &lt;160&gt; 86 &lt;170&gt; PatentIn, version 3.3 &lt;210&gt; 1 &lt;211&gt; 144 &lt; 212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic construct &lt;400&gt; 1

<td colspan="2">Met Trp 1</td><td>Lion</td><td>Gin</td><td>Cheese five</td><td>Lion</td><td colspan="3">Leu Leu Leu</td><td>Gly ten</td><td>Thr</td><td>hr</td><td>ala</td><td>cys</td><td>Cheese 15</td><td>how much</td>

<td>Cheese</td><td>ala</td><td>pro</td><td>ala</td><td rowspan="2">Angry</td><td>Cheese</td><td>Pro</td><td>Cheese</td><td>Pro</td><td>Cheese</td><td>Thr</td><td>Gin</td><td>Pro</td><td>Trp</td><td>Glu</td><td>His</td>

<td></td><td></td><td></td><td>20</td><td></td><td></td><td></td><td>25</td><td></td><td></td><td></td><td></td><td>thirty</td><td></td><td></td>

<td>hr</td><td>own</td><td>ala</td><td>How much</td><td>Gin</td><td>Glu</td><td>ala</td><td>Angry</td><td rowspan="2">Angry</td><td>Lion</td><td>Lion</td><td>own</td><td>Lion</td><td>Cheese</td><td rowspan="2">Angry</td><td rowspan="2">Asp</td>

<td></td><td></td><td>35</td><td></td><td></td><td></td><td></td><td>40</td><td></td><td></td><td></td><td>45</td><td></td>

<td>Thr</td><td>ala</td><td>ala</td><td>Glu</td><td>Underworld</td><td>own</td><td>Glu</td><td>Thr</td><td>hr</td><td>Glu</td><td>hr</td><td>How much</td><td>Cheese</td><td>Glu</td><td>Underworld</td><td>phe</td>

<td></td><td>50</td><td></td><td></td><td></td><td></td><td>55</td><td></td><td></td><td></td><td></td><td>60</td><td></td><td></td><td></td><td></td>

<td>Asp</td><td>Lion</td><td>Gin</td><td>Glu</td><td>Pro</td><td>Thr</td><td rowspan="2">Cys</td><td>Lion</td><td>Gin</td><td>Thr</td><td>Angry</td><td>Lion</td><td>Glu</td><td>Lion</td><td rowspan="2">Tyr</td><td>list</td>

<td>65</td><td></td><td></td><td></td><td></td><td>70</td><td></td><td></td><td></td><td>75</td><td></td><td></td><td></td><td>80</td>

<td>Gin</td><td>Gly</td><td>Lion</td><td>Angry</td><td>Gly 85</td><td>Cheese</td><td>Lion</td><td>Thr</td><td>list</td><td>Lion 90</td><td>list</td><td>Gly</td><td>pro</td><td>Lion</td><td>Thr 95</td><td>Underworld</td>

<td>Underworld</td><td>ala</td><td>Cheese</td><td>His</td><td rowspan="2">Tyr</td><td rowspan="2">list</td><td>Gin</td><td>His</td><td>cys</td><td>Pro</td><td>Pro</td><td>Thr</td><td>Pro</td><td>Glu</td><td>Thr</td><td>Cheese</td>

<td></td><td></td><td></td><td>100</td><td></td><td></td><td>105</td><td></td><td></td><td></td><td></td><td>110</td><td></td><td></td>

Cys Ala Thr Gln Ile Ile Thr Phe Glu Being Phe Lys Glu Asn Leu Lys

115

120

125

Asp Phe Leu Leu val Ile Pro Phe Asp Cys Trp Glu Pro val Gln Glu 130 135 140 &lt;210&gt; 2 &lt;211&gt; 127 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic construct &lt;400&gt; 2

<td colspan="3" rowspan="2">Ala Pro Ala 1</td><td rowspan="2">Angry</td><td colspan="8">For Pro Cheese Ser Thr Gin Pro</td><td rowspan="2">Trp</td><td rowspan="2">Glu</td><td rowspan="2">His 15</td><td rowspan="2">hr</td>

<td colspan="3">five</td><td colspan="5">ten</td>

<td>own</td><td>ala</td><td>How much</td><td>Gin</td><td>Glu</td><td>ala</td><td rowspan="2">Angry</td><td rowspan="2">Angry</td><td>Lion</td><td>Lion</td><td>own</td><td>Lion</td><td>Cheese</td><td>Angry</td><td>Asp</td><td>Thr</td>

<td></td><td></td><td></td><td>20</td><td></td><td></td><td>25</td><td></td><td></td><td></td><td></td><td>thirty</td><td></td><td></td>

<td>ala</td><td>ala</td><td>Glu</td><td>Underworld</td><td>own</td><td>Glu</td><td>Thr</td><td>hr</td><td>Glu</td><td>hr</td><td>how much</td><td>Cheese</td><td>Glu</td><td>Underworld</td><td>phe</td><td rowspan="2">Asp</td>

<td></td><td></td><td>35</td><td></td><td></td><td></td><td></td><td>40</td><td></td><td></td><td></td><td></td><td>45</td><td></td><td></td>

<td>Lion</td><td>Gin</td><td>Glu</td><td>Pro</td><td>Thr</td><td rowspan="2">cys</td><td>Lion</td><td>Gin</td><td>Thr</td><td rowspan="2">Angry</td><td>Lion</td><td>Glu</td><td>Lion</td><td rowspan="2">Tyr</td><td rowspan="2">list</td><td>Gl n</td>

<td></td><td>50</td><td></td><td></td><td></td><td>55</td><td></td><td></td><td></td><td>60</td><td></td><td></td>

<td>Gly</td><td>Lion</td><td rowspan="2">Angry</td><td rowspan="2">Gly</td><td>Cheese</td><td>Lion</td><td>Thr</td><td rowspan="2">list</td><td>Lion</td><td rowspan="2">list</td><td>Gly</td><td>Pro</td><td>Lion</td><td>Thr</td><td>Underworld</td><td>Underworld</td>

<td>65</td><td></td><td></td><td>70</td><td></td><td></td><td>75</td><td></td><td></td><td></td><td></td><td>80</td>

<td>ala</td><td>Cheese</td><td>His</td><td rowspan="2">Tyr</td><td>list</td><td>Gin</td><td>His</td><td rowspan="2">cys</td><td>Pro</td><td>Pro</td><td>Thr</td><td>Pro</td><td>Glu</td><td>Thr</td><td>Cheese</td><td rowspan="2">Cys</td>

<td></td><td></td><td></td><td>85</td><td></td><td></td><td></td><td>90</td><td></td><td></td><td></td><td></td><td>95</td>

<td>Al a</td><td>Thr</td><td>Gin</td><td>how much</td><td>How much</td><td>Thr</td><td>phe</td><td>Glu</td><td>Cheese</td><td>phe</td><td rowspan="2">list</td><td>Glu</td><td>own</td><td>Lion</td><td>list</td><td rowspan="2">Asp</td>

<td></td><td></td><td></td><td>100</td><td></td><td></td><td></td><td></td><td>105</td><td></td><td></td><td></td><td>110</td><td>"</td>

<td>phe</td><td>Lion</td><td>Lion</td><td>hr</td><td>How much</td><td>Pro</td><td>phe</td><td>ASp</td><td rowspan="2">Cys</td><td rowspan="2">Trp</td><td>Glu</td><td>Pro</td><td>hr</td><td>Gin</td><td>Glu</td><td></td>

<td></td><td></td><td>115</td><td></td><td></td><td></td><td></td><td>120</td><td></td><td></td><td>125</td><td></td><td></td><td></td>

&lt;210&gt; 3 &lt;211&gt; 15 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic construct &lt;400&gt; 3

Glu His Val Asn Ala how much Gln Glu Ala Arg Arg Leu Leu Asn Leu 15 10 15 &lt;210&gt; 4 &lt;211&gt; 15 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic construct &lt;400&gt; 4

Thr Gln Pro Pro Trp Glu Sera Val Asn Ala Ile Gln Glu Ala Arg 15 10 15 &lt;210&gt; 5 &lt;211&gt; 15 &lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt;

&lt;223&gt; Synthetic construct &lt;400&gt; 5

Met Ala Ser Ser Tyr Lys Gln His Cys Pro Pro Thr Pro Glu Thr 15 10 15 &lt;210&gt; 6 &lt;211&gt; 47 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic construct &lt;400&gt; 6

<td>Lion</td><td>Cheese</td><td>Thr</td><td>ala</td><td>hr</td><td rowspan="2">list</td><td>Glu</td><td>Lion</td><td>hr</td><td>Glu</td><td>own</td><td>Cheese</td><td>Lion</td><td rowspan="2">Asp</td><td>ala</td><td rowspan="2">Gly</td>

<td>one</td><td></td><td></td><td></td><td>five</td><td></td><td></td><td></td><td>ten</td><td></td><td></td><td></td><td>15</td>

<td>ala</td><td>Thr</td><td>own</td><td>How much 20</td><td>ASp</td><td>Lion</td><td>list</td><td>Lion</td><td>list 25</td><td>Asp</td><td>Tyr</td><td>Gly</td><td>hr</td><td>Asp thirty</td><td>Lion</td><td>How much</td>

<td>Glu</td><td>hr</td><td>Cheese</td><td rowspan="2">Asp</td><td>own</td><td rowspan="2">Gly</td><td rowspan="2">cys</td><td>Gly</td><td>hr</td><td>Glu</td><td>Glu</td><td>Glu</td><td>own</td><td>phe</td><td>Glu</td><td></td>

<td></td><td></td><td>35</td><td></td><td>40</td><td></td><td></td><td></td><td></td><td>45</td><td></td><td></td><td></td>

&lt;210&gt; 7 &lt;211&gt; 50 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic construct

Leu Arg Gln wave Leu Cheese Asn Leu Leu Asp Asn Ala Ile Lys Tyr Thr 1 5 10 15 &lt;400&gt; 7

Pro Glu Gly Gly Glu Ile Thr Val Ser Leu Glu Arg Asp Gly Asp His 20 25 30

Leu Glu how Thr val Glu Asp Asn Gly Pro Gly how much Pro Glu Glu Asp 35 40 45

Leu Glu 50 &lt;210&gt; 8 &lt;211&gt; 451 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic construct &lt;400&gt; 8

<td>Glu one</td><td>hr</td><td>Gin</td><td>Lion</td><td>hr five</td><td>Glu</td><td>Cheese</td><td>Gly</td><td>Gly</td><td>Gly ten</td><td>Lion</td><td>hr</td><td>Gin</td><td>Pro</td><td>Gly 15</td><td>Gly</td>

<td>Cheese</td><td>Lion</td><td>Angry</td><td>Lion</td><td>Cheese</td><td rowspan="2">cys</td><td>ala</td><td>ala</td><td>Cheese</td><td rowspan="2">Gly</td><td>phe</td><td>Thr</td><td>phe</td><td>Cheese</td><td rowspan="2">Angry</td><td>His</td>

<td></td><td></td><td></td><td>20</td><td></td><td></td><td></td><td>25</td><td></td><td></td><td></td><td>thirty</td><td></td>

<td>Trp</td><td>Underworld</td><td>His 35</td><td>Trp</td><td>Lion</td><td>Angry</td><td>Gin</td><td>hr 40</td><td>Pro</td><td>Gly</td><td>list</td><td>Gly</td><td>Pro 45</td><td>hr</td><td>Trp</td><td>hr</td>

<td>Cheese</td><td>Angry</td><td>how much</td><td>own</td><td rowspan="2">Gly</td><td>ala</td><td>Gly</td><td>Thr</td><td>Cheese</td><td>How much</td><td>Thr</td><td>Tyr</td><td>ala</td><td rowspan="2">Asp</td><td>Cheese</td><td>hr</td>

<td></td><td>50</td><td></td><td></td><td></td><td>55</td><td></td><td></td><td></td><td></td><td>60</td><td></td><td></td><td></td>

<td>Angry</td><td rowspan="2">Gly</td><td rowspan="2">Angry</td><td>phe</td><td>Thr</td><td>how much</td><td>Cheese</td><td rowspan="2">Angry</td><td rowspan="2">Asp</td><td>own</td><td>ala</td><td>own</td><td>own</td><td>Thr</td><td>Lion</td><td>phe</td>

<td>65</td><td></td><td></td><td>70</td><td></td><td></td><td>75</td><td></td><td></td><td></td><td></td><td>80</td>

<td>Lion</td><td>Gin</td><td>Underworld</td><td>own</td><td>Cheese</td><td>Lion</td><td>Angry</td><td>ala</td><td>Asp</td><td>Asp</td><td>Thr</td><td>ala</td><td>Lion</td><td rowspan="2">Tyr</td><td>phe</td><td rowspan="2">Cys</td>

<td></td><td></td><td></td><td></td><td>85</td><td></td><td></td><td></td><td></td><td>90</td><td></td><td></td><td></td><td>95</td>

<td>ala</td><td>Angry</td><td>ala</td><td>own 100</td><td>cheese</td><td>hr</td><td>Trp</td><td>phe</td><td>Angry 105</td><td>Gly</td><td>Lion</td><td>phe</td><td>Asp</td><td>Tyr 110</td><td>Trp</td><td>Gly</td>

<td>Gln</td><td rowspan="2">Gly</td><td>Thr</td><td>Pro</td><td>hr</td><td>Thr</td><td>hr</td><td>Cheese</td><td>Cheese</td><td>ala</td><td>Cheese</td><td>Thr</td><td>list</td><td rowspan="2">Gly</td><td>Pro</td><td>Cheese</td>

<td></td><td>115</td><td></td><td></td><td></td><td></td><td>120</td><td></td><td></td><td></td><td></td><td>125</td><td></td><td></td>

<td>hr</td><td>phe</td><td>Pro</td><td>Lion</td><td>ala</td><td>Pro</td><td>Cheese</td><td>Cheese</td><td rowspan="2">list</td><td>Cheese</td><td>Thr</td><td>Cheese</td><td rowspan="2">Gly</td><td rowspan="2">Gly</td><td>Thr</td><td>ala</td>

<td></td><td>130</td><td></td><td></td><td></td><td></td><td>135</td><td></td><td></td><td></td><td>140</td><td></td><td></td>

<td>ala</td><td>Lion</td><td rowspan="2">Gly</td><td rowspan="2">Cys</td><td>Lion</td><td>hr</td><td>list</td><td>Asp</td><td>Tyr</td><td>phe</td><td>Pro</td><td>Glu</td><td>Pro</td><td>hr</td><td>Thr</td><td>hr</td>

<td>145</td><td></td><td></td><td>150</td><td></td><td></td><td></td><td></td><td>155</td><td></td><td></td><td></td><td></td><td>160</td>

<td colspan="2" rowspan="2">Trp cheese</td><td rowspan="2">own</td><td rowspan="2">Cheese</td><td colspan="7">Gly Ala Leu Thr Ser Gly Val</td><td rowspan="2">His</td><td rowspan="2">Thr</td><td rowspan="2">phe</td><td rowspan="2">Pro 175</td><td rowspan="2">ala</td>

<td>165</td><td colspan="6">170</td>

<td>hr</td><td>Lion</td><td>Gln</td><td>Cheese</td><td>Cheese</td><td rowspan="2">Gly</td><td>Lion</td><td rowspan="2">Tyr</td><td>Cheese</td><td>Lion</td><td>Cheese</td><td>Cheese</td><td>hr</td><td>hr</td><td>Thr</td><td>hr</td>

<td></td><td></td><td></td><td>180</td><td></td><td></td><td>185</td><td></td><td></td><td></td><td></td><td>190</td><td></td><td></td>

<td>Pro</td><td>Cheese</td><td>Cheese</td><td>Cheese</td><td>Lion</td><td rowspan="2">Gly</td><td>Thr</td><td>Gin</td><td>Thr</td><td rowspan="2">Tyr</td><td>How much</td><td rowspan="2">cys</td><td>own</td><td>hr</td><td>own</td><td>His</td>

<td></td><td></td><td>195</td><td></td><td></td><td></td><td>200</td><td></td><td></td><td>205</td><td></td><td></td><td></td>

<td rowspan="2">list</td><td>Pro</td><td>Cheese</td><td>own</td><td>Thr</td><td rowspan="2">list</td><td>hr</td><td>Asp</td><td>list</td><td>list</td><td>hr</td><td>Glu</td><td>Pro</td><td>list</td><td>Cheese</td><td>cys</td>

<td>210</td><td></td><td></td><td></td><td>215</td><td></td><td></td><td></td><td></td><td>220</td><td></td><td></td><td></td><td></td>

<td>ASp</td><td rowspan="2">list</td><td>Thr</td><td>Hi s</td><td>Thr</td><td>cys</td><td>pro</td><td>Pro</td><td>Cys</td><td>Pro</td><td>ala</td><td>Pro</td><td>Glu</td><td>Lion</td><td>Lion</td><td>Gly</td>

<td>225</td><td></td><td></td><td></td><td>230</td><td></td><td></td><td></td><td></td><td>235</td><td></td><td></td><td></td><td></td><td>240</td>

<td rowspan="2">Gly</td><td>Pro</td><td>Cheese</td><td>hr</td><td>phe</td><td>Lion</td><td>phe</td><td>Pro</td><td>Pro</td><td>list</td><td>Pro</td><td rowspan="2">list</td><td rowspan="2">Asp</td><td>Thr</td><td>Lion</td><td>Underworld</td>

<td></td><td></td><td></td><td>245</td><td></td><td></td><td></td><td></td><td>250</td><td></td><td></td><td>255</td><td></td>

<td>How much</td><td>Cheese</td><td rowspan="2">Angry</td><td>Thr</td><td>Pro</td><td>Glu</td><td>hr</td><td>Thr</td><td>cys</td><td>hr</td><td>hr</td><td>hr</td><td rowspan="2">Asp</td><td>hr</td><td>Cheese</td><td>His</td>

<td></td><td></td><td>260</td><td></td><td></td><td></td><td></td><td>265</td><td></td><td></td><td></td><td>270</td><td></td><td></td>

<td>Glu</td><td rowspan="2">ASp</td><td>Pro</td><td>Glu</td><td>hr</td><td rowspan="2">list</td><td>phe</td><td>own</td><td>Trp</td><td>Tyr</td><td>hr</td><td>Asp</td><td>Gly</td><td>hr</td><td>Glu</td><td>hr</td>

<td></td><td>275</td><td></td><td></td><td></td><td>280</td><td></td><td></td><td></td><td></td><td>285</td><td></td><td></td><td></td>

<td>His</td><td>own</td><td>ala</td><td rowspan="2">list</td><td>Thr</td><td rowspan="2">list</td><td>Pro</td><td rowspan="2">Angry</td><td>Glu</td><td>Glu</td><td>Gin</td><td>Tyr</td><td>own</td><td>Cheese</td><td>Thr</td><td rowspan="2">Tyr</td>

<td></td><td>290</td><td></td><td></td><td>295</td><td></td><td></td><td></td><td>300</td><td></td><td></td><td></td>

<td>Angry</td><td>hr</td><td>hr</td><td>Cheese</td><td>hr</td><td>Lion</td><td>Thr</td><td>hr</td><td>Lion</td><td>His</td><td>Gin</td><td rowspan="2">Asp</td><td rowspan="2">Trp</td><td>Lion</td><td>own</td><td>Gly</td>

<td>305</td><td></td><td></td><td></td><td></td><td>310</td><td></td><td></td><td></td><td></td><td>315</td><td></td><td></td><td>320</td>

<td rowspan="2">list</td><td>Glu</td><td rowspan="2">Tyr</td><td rowspan="2">list</td><td>Cys</td><td rowspan="2">list</td><td>hr</td><td>Cheese</td><td>own</td><td>list</td><td>ala</td><td>Lion</td><td>Pro</td><td>ala</td><td>Pro</td><td>how much</td>

<td></td><td>325</td><td></td><td></td><td></td><td>330</td><td></td><td></td><td></td><td></td><td>335 *</td><td></td>

<td>Glu</td><td rowspan="2">list</td><td>Thr</td><td>how much</td><td>Cheese</td><td rowspan="2">list</td><td>ala</td><td rowspan="2">list</td><td>Gly</td><td>Gin</td><td>Pro</td><td rowspan="2">Angry</td><td>Glu</td><td>Pro</td><td>Gin</td><td>hr</td>

<td></td><td></td><td>340</td><td></td><td></td><td>345</td><td></td><td></td><td></td><td>350</td><td></td><td></td>

<td rowspan="2">Tyr</td><td>Thr</td><td>Lion</td><td>Pro</td><td>Pro</td><td>Cheese</td><td rowspan="2">Angry</td><td>ASp</td><td>Glu</td><td>Lion</td><td>Thr</td><td rowspan="2">list</td><td>own</td><td>Gin</td><td>hr</td><td>cheese</td>

<td></td><td>355</td><td></td><td></td><td></td><td>360</td><td></td><td></td><td></td><td>365</td><td></td><td></td><td></td>

<td>Lion</td><td>Thr</td><td rowspan="2">Cys</td><td>Lion</td><td>hr</td><td rowspan="2">list</td><td>Gly</td><td>phe</td><td rowspan="2">Tyr</td><td>Pro</td><td>Cheese</td><td>ASp</td><td>How much</td><td>ala</td><td>hr</td><td>Glu</td>

<td></td><td>370</td><td></td><td></td><td>375</td><td></td><td></td><td></td><td>380</td><td></td><td></td><td></td><td></td>

<td>Trp</td><td>Glu</td><td>Cheese</td><td>own</td><td rowspan="2">Gly</td><td>Gin</td><td>pro</td><td>Glu</td><td>own</td><td>own</td><td>Tyr</td><td>list</td><td>Thr</td><td>Thr</td><td>Pro</td><td>Pro</td>

<td>385</td><td></td><td></td><td></td><td>390</td><td></td><td></td><td></td><td></td><td>395</td><td></td><td></td><td></td><td></td><td>400</td>

<td>hr</td><td>Lion</td><td>Asp</td><td>Cheese</td><td>Asp 405</td><td>two</td><td>Cheese</td><td>phe</td><td>phe</td><td>Lion 410</td><td>Tyr</td><td>Cheese</td><td>list</td><td>Lion</td><td>Thr 415</td><td>hr</td>

<td rowspan="2">ASp</td><td rowspan="2">list</td><td>Cheese</td><td colspan="3">Arg Trp Gin</td><td>Gin</td><td rowspan="2">Gly</td><td>own</td><td>hr</td><td>phe</td><td colspan="2">Cys cheese</td><td>Cheese</td><td>hr</td><td>Underworld</td>

<td></td><td>420</td><td></td><td></td><td></td><td>425</td><td></td><td></td><td></td><td></td><td>430</td><td></td><td></td>

<td>His</td><td>Glu</td><td>ala</td><td>Lion</td><td>His</td><td>own</td><td>His</td><td>Tyr</td><td>Thr</td><td>Gin</td><td rowspan="2">list</td><td>Cheese</td><td>Lion</td><td>Cheese</td><td>Lion</td><td>Cheese</td>

<td></td><td></td><td>435</td><td></td><td></td><td></td><td></td><td>440</td><td></td><td></td><td></td><td>445</td><td></td><td></td><td></td>

Pro Gly Lys 450 &lt;210&gt; 9 &lt;211&gt; 470 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic construct &lt;400&gt; 9

<td colspan="2" rowspan="2">Met Gly 1</td><td rowspan="2">Trp</td><td rowspan="2">Cheese</td><td rowspan="2">Cys five</td><td colspan="2" rowspan="2">With</td><td colspan="8">Leu Phe Leu val Ala Thr Ala Thr</td><td rowspan="2">Gly</td>

<td colspan="3">ten</td><td colspan="5">15</td>

<td>hr</td><td>Hi 5</td><td>Cheese</td><td>Glu</td><td>hr</td><td>Gin</td><td>Lion</td><td>hr</td><td>Glu</td><td>Cheese</td><td rowspan="2">Gly</td><td rowspan="2">Gly</td><td rowspan="2">Gly</td><td>Lion</td><td>hr</td><td>Gin</td>

<td></td><td></td><td></td><td>20</td><td></td><td></td><td></td><td></td><td>25</td><td></td><td>thirty</td><td></td><td></td>

<td>pro</td><td colspan="2">Gly Gly</td><td>Cheese</td><td>Lion</td><td rowspan="2">Angry</td><td>Lion</td><td>Cheese</td><td rowspan="2">Cys</td><td>ala</td><td>ala</td><td>Cheese</td><td>Gly</td><td>phe</td><td>Thr</td><td>phe</td>

<td></td><td></td><td>35</td><td></td><td></td><td></td><td>40</td><td></td><td></td><td></td><td>45</td><td></td><td></td><td></td>

<td>Cheese</td><td>Angry 50</td><td>His</td><td>Trp</td><td>Underworld</td><td>His</td><td>Trp 55</td><td>Lion</td><td>Angry</td><td>Gin</td><td>hr</td><td>Pro 60</td><td>Gly</td><td>list</td><td>Gly</td><td>Pro</td>

<td>hr 65</td><td>Trp</td><td>hr</td><td>Cheese</td><td>Angry</td><td>How much 70</td><td>own</td><td>Gly</td><td>ala</td><td>Gly</td><td>Thr 75</td><td>Cheese</td><td>How much</td><td>Thr</td><td>Tyr</td><td>ala 80</td>

<td rowspan="2">Asp</td><td>Cheese</td><td>hr</td><td rowspan="2">Angry</td><td>Gly</td><td>Angry</td><td>phe</td><td>Thr</td><td>how much</td><td>Cheese</td><td rowspan="2">Angry</td><td>Asp</td><td>own</td><td>ala</td><td>own</td><td>own</td>

<td></td><td></td><td>85</td><td></td><td></td><td></td><td></td><td>90</td><td></td><td></td><td></td><td>95</td><td></td>

<td>Thr</td><td>Lion</td><td>phe</td><td>Lion</td><td>Gln</td><td>Underworld</td><td>own</td><td>Cheese</td><td>Lion</td><td rowspan="2">Angry</td><td>ala</td><td rowspan="2">ASp</td><td rowspan="2">ASp</td><td>Thr</td><td>ala</td><td>Lion</td>

<td></td><td></td><td></td><td>100</td><td></td><td></td><td></td><td></td><td>105</td><td></td><td>110</td><td></td><td></td>

<td rowspan="2">Tyr</td><td>phe</td><td>cys</td><td>ala</td><td>Angry</td><td>ala</td><td>own</td><td>Cheese</td><td>hr</td><td>Trp</td><td>phe</td><td>Angry</td><td>Gly</td><td>Lion</td><td>phe</td><td rowspan="2">Asp</td>

<td></td><td>115</td><td></td><td></td><td></td><td></td><td>120</td><td></td><td></td><td></td><td></td><td>125</td><td></td><td></td>

<td rowspan="2">Tyr</td><td>Trp</td><td rowspan="2">Gly</td><td>Gin</td><td>Gly</td><td>Thr</td><td>Pro</td><td>hr</td><td>Thr</td><td>hr</td><td>Cheese</td><td>Cheese</td><td>ala</td><td>Cheese</td><td>Thr</td><td rowspan="2">list</td>

<td>130</td><td></td><td></td><td></td><td>135</td><td></td><td></td><td></td><td></td><td>140</td><td></td><td></td><td></td>

<td>Gly</td><td>Pro</td><td>Cheese</td><td>hr</td><td>phe</td><td>Pro</td><td>Lion</td><td>ala</td><td>Pro</td><td>Cheese</td><td>Cheese</td><td>list</td><td>Cheese</td><td>Thr</td><td>Cheese</td><td>Gly</td>

<td>145</td><td></td><td></td><td></td><td></td><td>150</td><td></td><td></td><td></td><td></td><td>155</td><td></td><td></td><td></td><td></td><td>160</td>

<td>Gly</td><td>Thr</td><td>ala</td><td>ala</td><td>Lion 165</td><td>Gly</td><td>Cys</td><td>Lion</td>

<td>hr</td><td>Thr</td><td>VA1</td><td>Cheese 180</td><td>Trp</td><td>own</td><td>Cheese</td><td>Gly</td>

<td>phe</td><td>Pro</td><td>ala 195</td><td>hr</td><td>Lion</td><td>Gin</td><td>Cheese</td><td>Cheese 200</td>

<td>hr</td><td>Thr 210</td><td>hr</td><td>Pro</td><td>Cheese</td><td>Cheese</td><td>Cheese 215</td><td>Lion</td>

<td>hr 225</td><td>own</td><td>His</td><td>list</td><td>Pro</td><td>Cheese 230</td><td>own</td><td>Thr</td>

<td>list</td><td>Cheese</td><td>Cys</td><td>ASP</td><td>list 245</td><td>Thr</td><td>His</td><td>Thr</td>

<td>Lion</td><td>Lion</td><td colspan="2">Gly Gly 260</td><td>Pro</td><td>Cheese</td><td>hr</td><td>phe</td>

<td>Thr</td><td>Lion</td><td>Underworld 275</td><td>how much</td><td>Cheese</td><td>Angry</td><td>Thr</td><td>Pro 280</td>

<td>hr</td><td>Cheese 290</td><td>His</td><td>Glu</td><td>ASp</td><td>Pro</td><td>Glu 295</td><td>hr</td>

<td>hr 305</td><td>Glu</td><td>hr</td><td>His</td><td>own</td><td>ala 310</td><td>list</td><td>Thr</td>

<td>Cheese</td><td>Thr</td><td>Tyr</td><td>Angry</td><td>hr 325</td><td>hr</td><td>Cheese</td><td>hr</td>

<td>Lion</td><td>own</td><td>Gly</td><td>list 340</td><td>Glu</td><td>Tyr</td><td>list</td><td>Cys</td>

<td>ala</td><td>pro</td><td>How much 355</td><td>Glu</td><td>list</td><td>Thr</td><td>how much</td><td>Cheese 360</td>

<td>Pro</td><td>Gin 370</td><td>hr</td><td>Tyr</td><td>Thr</td><td>Lion</td><td>Pro 375</td><td>Pro</td>

<td>Gin 385</td><td>VA1</td><td>Cheese</td><td>Lion</td><td>Thr</td><td>cys 390</td><td>Lion</td><td>hr</td>

<td>ala</td><td>will!</td><td>Glu</td><td>Trp</td><td>Glu 405</td><td>Cheese</td><td>own</td><td>Gly</td>

<td>hr</td><td>list 170</td><td>Asp</td><td>Tyr</td><td>phe</td><td>Pro</td><td>Glu 175</td><td>pro</td>

<td>ala 185</td><td>Lion</td><td>Thr</td><td>Cheese</td><td>Gly</td><td>hr 190</td><td>His</td><td>Thr</td>

<td>Gly</td><td>Lion</td><td>Tyr</td><td>Cheese</td><td>Lion 205</td><td>Cheese</td><td>Cheese</td><td>hr</td>

<td>Gly</td><td>Thr</td><td>Gin</td><td>Thr 220</td><td>Tyr</td><td>How much</td><td>cys</td><td>own</td>

<td>list</td><td>hr</td><td>Asp 235</td><td>list</td><td>list</td><td>hr</td><td>Glu</td><td>Pro 240</td>

<td>cys</td><td>Pro 250</td><td>Pro</td><td>cys</td><td>pro</td><td>ala</td><td>Pro 255</td><td>Glu</td>

<td>Lion 265</td><td>phe</td><td>Pro</td><td>pro</td><td>list</td><td>Pro 270</td><td>list</td><td>Asp</td>

<td>Glu</td><td>hr</td><td>Thr</td><td>cys</td><td>hr 285</td><td>hr</td><td>hr</td><td>Asp</td>

<td>list</td><td>phe</td><td>own</td><td>Trp 300</td><td>Tyr</td><td>hr</td><td>ASp</td><td>Gly</td>

<td>list</td><td>Pro</td><td>Angry 315</td><td>Glu</td><td>Glu</td><td>Gin</td><td>Tyr</td><td>own 320</td>

<td>Lion</td><td>Thr 330</td><td>hr</td><td>Lion</td><td>His</td><td>Gin</td><td>ASp 335</td><td>Trp</td>

<td>list 345</td><td>hr</td><td>Cheese</td><td>own</td><td>list</td><td>ala 350</td><td>Lion</td><td>Pro</td>

<td>list</td><td>ala</td><td>list</td><td>Gly</td><td>Gin 365</td><td>Pro</td><td>Angry</td><td>Glu</td>

<td>Cheese</td><td>Angry</td><td>ASp</td><td>Glu 380</td><td>Lion</td><td>Thr</td><td>list</td><td>own</td>

<td>list</td><td>Gly</td><td>phe 395</td><td>Tyr</td><td>pro</td><td>Cheese</td><td>Asp</td><td>How much 400</td>

<td>Gln</td><td>Pro 410</td><td>Glu</td><td>own</td><td>own</td><td>Tyr</td><td>list 415</td><td>Thr</td>

<td>Thr</td><td>Pro</td><td>pro</td><td>hr</td><td>Lion</td><td rowspan="2">Asp</td><td>Cheese</td><td rowspan="2">Asp</td><td>Gly</td><td>Cheese</td><td>phe</td><td>phe</td><td>Lion</td><td>Tyr</td><td>cheese</td><td>list</td>

<td></td><td></td><td></td><td>420</td><td></td><td></td><td>425</td><td></td><td></td><td></td><td></td><td>430</td><td></td><td></td>

<td>Lion</td><td>Thr</td><td>hr</td><td rowspan="2">Asp</td><td rowspan="2">list</td><td>Cheese</td><td rowspan="2">Angry</td><td>Trp</td><td>Gin</td><td>Gin</td><td rowspan="2">Gly</td><td>own</td><td>hr</td><td>phe</td><td>Cheese</td><td>cys</td>

<td></td><td></td><td>435</td><td></td><td>440</td><td></td><td></td><td></td><td>445</td><td></td><td></td><td></td>

<td>Cheese</td><td>hr</td><td>Underworld</td><td>His</td><td>Glu</td><td>ala</td><td>Lion</td><td>His</td><td>own</td><td>Hi s</td><td rowspan="2">Tyr</td><td>Thr</td><td>Gin</td><td rowspan="2">list</td><td>Cheese</td><td>Lion</td>

<td></td><td>450</td><td></td><td></td><td></td><td></td><td>455</td><td></td><td></td><td></td><td>460</td><td></td><td></td><td></td>

<td>Cheese</td><td>Lion</td><td>Cheese</td><td>Pro</td><td>Gly</td><td>list</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

465 470 &lt;210&gt; 10 &lt;211&gt; 1356 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; oligonucleotide primer &lt;400&gt; 10

<td>gaggtgcagc tggtggagtc tgggggaggc ttagttcagc cgggggggtc cctgagactc</td><td>60</td>

<td>tcctgtgcag cctctggatt cactttcagt agacactgga tgcactggct tcgccaggtt</td><td>120</td>

<td>ccaggtaagg ggccggtctg ggtctcacgt atcaatggtg ctgggacttc cataacctac</td><td>180</td>

<td>gcggactccg tgaggggccg attcaccatc tccagagaca acgccaacaa cacactgttt</td><td>240</td>

<td>ctgcaaatga acagtctgag agccgacgac acggctcttt atttctgtgc aagagcgaac</td><td>. 300</td>

<td>agcgtctggt tccggggcct ctttgactac tggggccagg gaaccccggt caccgtctcc</td><td>360</td>

<td>tcagcctcca ccaagggccc atcggtcttc cccctggcac cctcctccaa gagcacctct</td><td>420</td>

<td>gggggcacag cggccctggg ctgcctggtc aaggactact tccccgaacc ggtgacggtg</td><td>480</td>

<td>tcgtggaact caggcgccct gaccagcggc gtgcacacct tcccggctgt cctacagtcc</td><td>540</td>

<td>tcaggactct actccctcag cagcgtggtg accgtgccct ccagcagctt gggcacccag</td><td>600</td>

<td>acctacatct gcaacgtgaa tcacaagccc agcaacacca aggtggacaa gaaagttgag</td><td>660</td>

<td>cccaaatctt gtgacaaaac tcacacatgc ccaccgtgcc cagcacctga actcctgggg</td><td>720</td>

<td>ggaccgtcag cctccctctt</td><td>780</td>

<td>cctgaggtca catgcgtggt ggtggacgtg agccacgaag accctgaggt caagttcaac</td><td>840</td>

<td>tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccgcggga ggagcagtac</td><td>900</td>

<td>aacagcacgt accgtgtggt cagcgtcctc accgtcctgc accaggactg gctgaatggc</td><td>960</td>

<td>glossary ctccaacaaa gccctcccag ccccatcatga gaaaaccatc</td><td>1020</td>

<td>tccaaagcca aagggcagcc ccgagaacca caggtgtaca ccctgccccc atcccgggat</td><td>1080</td>

<td>gagctgacca agaaccaggt cagcctgacc tgcctggtca aaggcttcta tcccagcgac</td><td>1140</td>

<td>atcgccgtgg agtgggagag caatgggcag ccggggaaca actacaagac cacgcctccc</td><td>1200</td>

<td>gtgctggact cgggcggctg cttcttcctc tacgcgaggc tcaccgtgga. ggggaggagg</td><td>1260</td>

<td>tggcggcggg gggacgtctt ctcatgctcc gttactcacg aggctctgca aaggcgctgc</td><td>1320</td>

<td>agggggaaga gggtctgcgt gtctcccggg aaacta</td><td>1356</td>

&lt;210&gt; 11 &lt;211&gt; 1434 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; An oligonucleotide primer &lt;400&gt; 11

<td>aagcttgccg ccaccatggg atggagctgt atcatcctct tcttggtagc aacagctaca</td><td>60</td>

<td>ggtgtacaca gcgaggtgca gctggtggag tctgggggag gcttagttca gccggggggg</td><td>120</td>

<td>tccctgagac tctcctgtgc agcctctgga ttcactttca gtagacactg gatgcactgg</td><td>180</td>

<td>cttcgccagg ttccaggtaa ggggccggtc tgggtctcac gtatcaatgg tgc ^ t ^^^ i ^ c ^ t</td><td>240</td>

<td>tccataacct -cccc-cic cgtgaggggc cgattcacca tctccagaga caacgccaac</td><td>300</td>

<td>aacacactgt ttctgcaate gaacagtctg agagccgacg acacggctct ttatttctgt</td><td>360</td>

<td>gcaagagcga acagcgtctg gttccggggc ctctttgact actggggcaca gggaaccccg</td><td>420</td>

<td>gtcaccgtct cctcagcctc caccaagggc ccatcggtct tccccctggc accctcctcc</td><td>480</td>

<td>aagagcacct «ccccc ^ c agcggccctg ggctgcctgg cttccccgaa</td><td>540</td>

<td>ccccIc-ccc tgtcgtcgaa ctcaggcgcc c-tgaccagcg gcg-tgcacac cttcccggct</td><td>600</td>

<td>gtcctacagt cctcagcact ctactccctc agcagcgtgg tgaccgtgcc ctccagcagc</td><td>660</td>

<td>ttgggcaccc agacctacat ctgcaacgtg ccagcaacac caaggtgcac</td><td>720</td>

<td>aagaaagttg agcccaaatc ttgtgacaaa acecacacat gcccccgtg cccagcacct</td><td>780</td>

<td>gaactcctgg gccgaccgtc agtcttcctc ftccccccaa aacccaagga caccctcatg</td><td>840</td>

<td>atctcccgga cccctgaggt cacatgcgtg gtggtggacg tgagccacga agaccctgag</td><td>900</td>

<td>gtcaagttca actggtacgt ggacggcgtg gaggtgcata atgccaagac aaagccgcgg</td><td>960</td>

<td>gagcagcagt acaacagcac gtaccgtgtg gtcagcgtcc tcaecgtccT caccaggac</td><td>1020</td>

<td>tggctgaatg tumblr bags</td><td>1080</td>

<td>gagaaaacca tctccaaagc caaagggcag ccccgagaac cacaggtgta caccctgccc</td><td>1140</td>

<td>ccatcccggg atgagctcac caagaaccag gtcagcctga cctgcctggt caaaggcttc</td><td>1200</td>

<td>tatcccagcg acatcgccgt ggagtgggag agcaatgggc agccggagaa caactacaag</td><td>1260</td>

<td>accacgcctc ccgtgctgga ctccgacggc tccttcftcc tctacagcaa gctcaccgtg</td><td>1320</td>

<td>gacaagagca ggtggcagca gcggaaccct ttctcatgct ccgtcatgca tgaggctctg</td><td>1380</td>

<td>cacaaccact acacacagaa gcgcctcccc ctgtctcccg gcaaatgaga attc</td><td>1434</td>

&lt;210&gt; 12 &lt;211&gt; 214 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic construct &lt;400&gt; 12

<td colspan="3">Val with Glu 1</td><td colspan="2">Leu Thr 5</td><td colspan="4">Gin Ser Pro v</td><td colspan="4">Thr Leu Be worth 10</td><td>Cheese</td><td>Pro 15</td><td>Gly</td>

<td>Glu</td><td rowspan="2">Angry</td><td>hr</td><td>Thr</td><td>Lion</td><td>Cheese</td><td rowspan="2">cys</td><td rowspan="2">Angry</td><td>ala</td><td>Cheese</td><td>Gin</td><td>Cheese</td><td>hr</td><td>Cheese</td><td>Thr</td><td>own</td>

<td></td><td></td><td>20</td><td></td><td></td><td>25</td><td></td><td></td><td></td><td></td><td>thirty</td><td></td><td></td>

<td>Lion</td><td>ala</td><td>Trp</td><td rowspan="2">Tyr</td><td>Gin</td><td>Gin</td><td rowspan="2">list</td><td>Lion</td><td>Gly</td><td>Gl n</td><td>Gly</td><td>Pro</td><td>Angry</td><td>Lion</td><td>Lion</td><td>how much</td>

<td></td><td></td><td>35</td><td></td><td></td><td>40</td><td></td><td></td><td></td><td></td><td>45</td><td></td><td></td><td></td>

<td rowspan="2">Tyr</td><td>Gly</td><td>ala</td><td>Cheese</td><td>Thr</td><td rowspan="2">Angry</td><td>ala</td><td>Thr</td><td rowspan="2">Asp</td><td>How much</td><td>Pro</td><td>ala</td><td rowspan="2">Angry</td><td>phe</td><td>Cheese</td><td rowspan="2">Gly</td>

<td>50</td><td></td><td></td><td></td><td>55</td><td></td><td></td><td></td><td>60</td><td></td><td></td>

<td>Cheese</td><td rowspan="2">Gly</td><td>Cheese</td><td>Glu</td><td>Thr</td><td>Glu</td><td>phe</td><td>Thr</td><td>Lion</td><td>Thr</td><td>how much</td><td>Cheese</td><td>Cheese</td><td>Lion</td><td>Gin</td><td>Cheese</td>

<td>65</td><td></td><td></td><td></td><td>70</td><td></td><td></td><td></td><td></td><td>75</td><td></td><td></td><td></td><td></td><td>80</td>

<td>Glu</td><td>Asp</td><td>phe</td><td>ala</td><td>hr</td><td>Tyr</td><td>Tyr</td><td>Cys</td><td>Gin</td><td>Gin</td><td>Tyr</td><td>Asp</td><td>list</td><td>Trp</td><td>Pro</td><td>Asp</td>

<td></td><td></td><td></td><td></td><td>85</td><td></td><td></td><td></td><td></td><td>90</td><td></td><td></td><td></td><td></td><td>95</td><td></td>

<td>Thr</td><td>phe</td><td>Gly</td><td>Gin 100</td><td>Gly</td><td>Thr</td><td>list</td><td>Lion</td><td>Glu 105</td><td>how much</td><td>list</td><td>Angry</td><td>Thr</td><td>hr 110</td><td>ala</td><td>ala</td>

<td>Pro</td><td>Cheese</td><td>hr</td><td>phe</td><td>how much</td><td>phe</td><td>Pro</td><td>Pro</td><td>Cheese</td><td rowspan="2">Asp</td><td>Glu</td><td>Gin</td><td>Lion</td><td rowspan="2">list</td><td>Cheese</td><td rowspan="2">Gly</td>

<td></td><td></td><td>115</td><td></td><td></td><td></td><td></td><td>120</td><td></td><td></td><td></td><td>125</td><td></td>

<td>Thr</td><td>ala</td><td>Cheese</td><td>hr</td><td>hr</td><td rowspan="2">cys</td><td>Lion</td><td>Lion</td><td>own</td><td>own</td><td>phe</td><td>Tyr</td><td>Pro</td><td rowspan="2">Angry</td><td>Glu</td><td>ala</td>

<td></td><td>130</td><td></td><td></td><td></td><td>135</td><td></td><td></td><td></td><td></td><td>140</td><td></td><td></td><td></td>

<td>list</td><td>hr</td><td>Gin</td><td rowspan="2">Trp</td><td rowspan="2">^Theys</td><td>hr</td><td rowspan="2">Asp</td><td>own</td><td>ala</td><td>Lion</td><td>Gin</td><td>Cheese</td><td rowspan="2">Gly</td><td>own</td><td>Cheese</td><td>Gin</td>

<td>145</td><td></td><td></td><td>150</td><td></td><td></td><td></td><td>155</td><td></td><td></td><td></td><td>160</td>

<td>Glu</td><td>Cheese</td><td>hr</td><td>Thr</td><td>Glu</td><td>Gin</td><td rowspan="2">Asp</td><td>Cheese</td><td rowspan="2">list</td><td>Asp</td><td>Cheese</td><td>Thr</td><td rowspan="2">Tyr</td><td>Cheese</td><td>Lion</td><td>Cheese</td>

<td></td><td></td><td></td><td></td><td>165</td><td></td><td></td><td>170</td><td></td><td></td><td></td><td>175</td><td></td>

<td>Cheese</td><td>Thr</td><td>Lion</td><td>Thr 180</td><td>Lion</td><td>Cheese</td><td>list</td><td>ala</td><td>Asp 185</td><td>Tyr</td><td>Glu</td><td>list</td><td>His</td><td>list 190</td><td>hr</td><td>Tyr</td>

<td colspan="2">Ala Cys</td><td>Glu</td><td>val</td><td>Thr</td><td>His</td><td>Gin</td><td>Gly</td><td>Leu</td><td>Cheese</td><td>Cheese</td><td colspan="2">Pro val</td><td>Thr</td><td>lys</td><td>cheese</td>

195,200 205

Phe Asn Arg Gly Glu Cys 210 &lt;210&gt; 13 &lt;211&gt; 233 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic build &lt;400&gt; 13

<td>Underworld 1</td><td>Gly</td><td colspan="2">Trp Ser</td><td colspan="5">Cys Ile how Leu Phe 5</td><td colspan="2">Leu val 10</td><td>ala</td><td>Thr</td><td>ala</td><td>Thr 15</td><td>Gly</td>

<td>val</td><td>His</td><td>Cheese</td><td>Glu</td><td>how much</td><td>val</td><td>Leu</td><td>Thr</td><td>Gln</td><td>Cheese</td><td>Pro</td><td>val</td><td>Thr</td><td>Leu</td><td>Cheese</td><td>val</td>

<td></td><td></td><td></td><td>20</td><td></td><td></td><td></td><td></td><td>25</td><td></td><td></td><td></td><td></td><td>thirty</td><td></td><td></td>

<td>Cheese</td><td>Pro</td><td>Gly</td><td>Glu</td><td rowspan="2">Arg</td><td>val</td><td>Thr</td><td>Leu</td><td>Cheese</td><td rowspan="2">cys</td><td rowspan="2">Arg</td><td>ala</td><td>Cheese</td><td>Gln</td><td>Cheese</td><td>val</td>

<td></td><td></td><td>35</td><td></td><td></td><td></td><td>40</td><td></td><td></td><td>45</td><td></td><td></td><td></td>

<td>Cheese</td><td>Thr</td><td>own</td><td>Leu</td><td>ala</td><td rowspan="2">Trp</td><td>Tyr</td><td>Gln</td><td>Gln</td><td>lys</td><td>Leu</td><td>Gly</td><td>Gln</td><td rowspan="2">Gly</td><td>Pro</td><td rowspan="2">Arg</td>

<td></td><td>50</td><td></td><td></td><td></td><td>55</td><td></td><td></td><td></td><td></td><td>60</td><td></td><td></td>

<td>Leu</td><td>Leu</td><td>How much</td><td rowspan="2">Tyr</td><td rowspan="2">Gly</td><td>ala</td><td>Cheese</td><td>Thr</td><td rowspan="2">Arg</td><td>ala</td><td>Thr</td><td>ASP</td><td>how much</td><td>Pro</td><td>ala</td><td>Arg</td>

<td>65</td><td></td><td></td><td>70</td><td></td><td></td><td></td><td>75</td><td></td><td></td><td></td><td></td><td>80</td>

<td>phe</td><td>Cheese</td><td rowspan="2">Gly</td><td>Cheese</td><td>Gl</td><td>Cheese</td><td>Glu</td><td>Thr</td><td>Glu</td><td>phe</td><td>Thr</td><td>Leu</td><td>Thr</td><td>how much</td><td>Cheese</td><td>Cheese</td>

<td></td><td></td><td></td><td>85</td><td></td><td></td><td></td><td></td><td>90</td><td></td><td></td><td></td><td></td><td>95</td><td></td>

<td>Leu</td><td>Gln</td><td>Cheese</td><td>Glu 100</td><td>ASP</td><td>phe</td><td>ala</td><td>val</td><td>Tyr 105</td><td>Tyr</td><td>cys</td><td>Gln</td><td>Gln</td><td>Tyr 110</td><td>Asp</td><td>lys</td>

<td rowspan="2">Trp</td><td>Pro</td><td>Asp</td><td>Thr</td><td>phe</td><td rowspan="2">Gly</td><td>Gln</td><td>Gly</td><td>Thr</td><td rowspan="2">lys</td><td>Leu</td><td>Glu</td><td>How much</td><td>lys</td><td>Arg</td><td>Thr</td>

<td></td><td>115</td><td></td><td></td><td></td><td>120</td><td></td><td></td><td></td><td>125</td><td></td><td></td><td></td>

<td>val</td><td>ala</td><td>ala</td><td>Pro</td><td>Cheese</td><td>val</td><td>phe</td><td>How much</td><td>phe</td><td>pro</td><td>pro</td><td>Cheese</td><td rowspan="2">ASP</td><td>Glu</td><td>Gln</td><td>Leu</td>

<td></td><td>130</td><td></td><td></td><td></td><td></td><td>135</td><td></td><td></td><td></td><td></td><td>140</td><td></td><td></td><td></td>

<td>lys 145</td><td>Cheese</td><td>Gly</td><td>Thr</td><td>ala</td><td>Cheese 150</td><td>val</td><td>val</td><td>Cys</td><td>Leu</td><td>Leu 155</td><td>own</td><td>own</td><td>phe</td><td>Tyr</td><td>Pro 160</td>

<td rowspan="2">Arg</td><td>Glu</td><td>ala</td><td rowspan="2">lys</td><td>val</td><td>Gln</td><td rowspan="2">Trp</td><td rowspan="2">lys</td><td>val</td><td>Asp</td><td>own</td><td>ala</td><td>Leu</td><td>Gln</td><td>Cheese</td><td rowspan="2">Gly</td>

<td></td><td></td><td>165</td><td></td><td></td><td>170</td><td></td><td></td><td></td><td></td><td>175</td>

<td>own</td><td>Cheese</td><td>Gln</td><td>Glu</td><td>Cheese</td><td>val</td><td>Thr</td><td>Glu</td><td>Gln</td><td>ASP</td><td>Cheese</td><td>lys</td><td>Asp</td><td>Cheese</td><td>Thr</td><td>Tyr</td>

<td></td><td></td><td></td><td>180</td><td></td><td></td><td></td><td></td><td>185</td><td></td><td></td><td></td><td></td><td colspan="2">190</td><td></td>

<td>Cheese</td><td>Leu</td><td>Cheese</td><td>Cheese</td><td>Thr</td><td>Leu</td><td>Thr</td><td>Leu</td><td>Cheese</td><td>lys</td><td colspan="2">Ala Asp</td><td>Tyr</td><td>Glu</td><td rowspan="2">lys</td><td>and His</td>

<td></td><td></td><td>195</td><td></td><td></td><td></td><td></td><td>200</td><td></td><td></td><td></td><td></td><td>205</td><td></td><td></td>

<td>lys</td><td>val 210</td><td>Tyr</td><td>ala</td><td>cys</td><td>Glu</td><td>val 215</td><td>Thr</td><td>His</td><td>Gln</td><td colspan="2">Gly Leu 220</td><td>Cheese</td><td>Cheese</td><td colspan="2">Pro val</td>

<td>Thr 225</td><td>lys</td><td>Cheese</td><td>phe</td><td>own</td><td></td><td>Gly</td><td>Glu</td><td>cys</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

&lt;210&gt; 14 &lt;211&gt; 645 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; oligonucleotide primer &lt;400&gt; 14 gaaattgtgc tgactcagtc tccagtcacc ctgtctgtgt ctccagggga aagagtcact 60 ctctcctgca gggccagtca gagtgttagc accaacttag cctggtatca gcagaaactt 120 ggccagggtc ccaggctcct catttatggt gcatccacca gggccactga tatcccagcc 180 aggttcagtg gcagtgggtc tgagacagag ttcactctca ccatcagcag cctgcagtct 240 gaagattttg cagtttatta ctgtcagcaa tatgataagt ggccggacac ttttggccag 300 gggaccaagc tggagatcaa acgaactgtg gctgcaccat ctgtcttcat cttcccgcca 360 tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat 420 cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag 480 gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg 540 ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ca ^ cagggc 600 ctgagttcgc ccgtcacaaa gagcttcaac aggggagagt GTTA 645 &lt;210&gt; 15 &lt;211&gt; 723 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; oligonucleotide primer &lt;400&gt; 15 aagcttgccg ccaccattgg atcatcctct tcttggtaac aacagctaca 60 ggtgtacaca gtagataccg gctgactcag tctccagtca cacactccgt grcrccaggg 120 ttatttttaa ccctctccac cagggccag-c cagagtgtta ggatcaactt aggctggtat 180 ttcggcatgg tgggagggtc ctcatttaag ggcgcttaac cattlgcgagt 240 gactcctcat tttgttcctt tgtgagtggg tctgagacag agttaaatat eaggatgagg 300 tgcacgttgt ttttagattt tgtactLt: this c Tagtgtgagc aacaagagaa 360 tgtgtaccta gggzggagaag aaaggaaerg tęgattgagc aactgtg-ttc 420 tccctctcgt tacctttttt gcagttgaaa GCT tctggaactg: cttt: CGT: 480 gtgggtggtg tatattccct tctttatata ggcaaaagta cattggaagg ccaccaarcg 540 ggtattctct ttttgagttt aacacagaag ggcatgaagg acagaaacta cagggtgagg 600 agtacactac gagaaagaca aattctcaat CT: gcgaagt: 660 g tttcgtgcct gggggtaaga aataacac: ga acagttttaJa gagraaagaa 720 ttc 723 &lt;210&gt; 16 &lt;211&gt;220 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;400&gt; 16 &lt;220&gt;

&lt;223&gt; Synthetic build

<td colspan="2" rowspan="2">Gln val 1</td><td rowspan="2">Gin</td><td colspan="2" rowspan="2">Leu Glu 5</td><td colspan="2" rowspan="2">Glu Cheese</td><td rowspan="2">Gly</td><td colspan="8">Gly Gly van Gin Pro Gly Arg</td>

<td colspan="5">10</td><td colspan="3">15</td>

<td>Cheese</td><td>Leu</td><td rowspan="2">Arg</td><td>Leu</td><td>Cheese</td><td rowspan="2">Cys</td><td>ala</td><td>ala</td><td>Cheese</td><td rowspan="2">Gly</td><td>phe</td><td>Thr</td><td>phe</td><td>Thr</td><td>own</td><td rowspan="2">Tyr</td>

<td></td><td></td><td>20</td><td></td><td></td><td></td><td>25</td><td></td><td></td><td></td><td>thirty</td><td></td>

<td rowspan="2">Gly</td><td>Underworld</td><td>His</td><td rowspan="2">Trp</td><td>val</td><td rowspan="2">Arg</td><td>Gin</td><td>ala</td><td>Pro</td><td>Gly</td><td>lys</td><td>Gly</td><td>Leu</td><td>Glu</td><td rowspan="2">Trp</td><td>Leu</td>

<td></td><td>35</td><td></td><td></td><td>40</td><td></td><td></td><td></td><td></td><td>45</td><td></td><td></td>

<td>ala</td><td>Leu</td><td>How much</td><td>Cheese</td><td rowspan="2">Tyr</td><td rowspan="2">Asp</td><td>Gly</td><td>own</td><td rowspan="2">Arg</td><td>Gin</td><td>Tyr</td><td>Tyr</td><td>ala</td><td rowspan="2">Asp</td><td>Cheese</td><td>val</td>

<td></td><td>50</td><td></td><td></td><td>55</td><td></td><td></td><td></td><td>60</td><td></td><td></td><td></td>

<td>lys</td><td rowspan="2">Gly</td><td rowspan="2">Arg</td><td>phe</td><td>Thr</td><td>val</td><td>Cheese</td><td rowspan="2">Arg</td><td rowspan="2">Asp</td><td>own</td><td>pro</td><td>own</td><td>own</td><td>Thr</td><td>Leu</td><td>His</td>

<td>65</td><td></td><td></td><td>70</td><td></td><td></td><td>75</td><td></td><td></td><td></td><td></td><td>80</td>

<td>Leu</td><td>Glu</td><td>Underworld</td><td rowspan="2">lys</td><td>Cheese</td><td>Leu</td><td rowspan="2">Arg</td><td>ala</td><td>Glu</td><td>Asp</td><td>Cheese</td><td>ala</td><td>how much</td><td rowspan="2">Tyr</td><td>Tyr</td><td rowspan="2">Cys</td>

<td></td><td></td><td></td><td>85</td><td></td><td></td><td></td><td>90</td><td></td><td></td><td></td><td>95</td>

<td>ala</td><td rowspan="2">Arg</td><td rowspan="2">Gly</td><td>ala</td><td rowspan="2">Gly</td><td>val</td><td>Leu</td><td>Leu</td><td>Trp</td><td>phe</td><td>Gly</td><td>Asp</td><td>Leu</td><td>Cheese</td><td>Trp</td><td>phe</td>

<td></td><td>100</td><td></td><td></td><td></td><td>105</td><td></td><td></td><td></td><td></td><td>110</td><td></td><td></td>

<td rowspan="2">ASP</td><td>Pro</td><td>Trp</td><td rowspan="2">Gly</td><td>Gin</td><td rowspan="2">Gly</td><td>Thr</td><td>Leu</td><td>val</td><td>Thr</td><td>val</td><td>Cheese</td><td>Cheese</td><td>ala</td><td>Cheese</td><td>Thr</td>

<td></td><td>115</td><td></td><td></td><td>120</td><td></td><td></td><td></td><td></td><td>125</td><td></td><td></td><td></td>

<td>lys</td><td>Gly</td><td>Pro</td><td>Cheese</td><td>val</td><td>phe</td><td>Pro</td><td>Leu</td><td>ala</td><td>Pro</td><td>Cheese</td><td>Cheese</td><td>lys</td><td>Cheese</td><td>Thr</td><td>Cheese</td>

<td></td><td>130</td><td></td><td></td><td></td><td></td><td>135</td><td></td><td></td><td></td><td></td><td>140</td><td></td><td></td><td></td><td></td>

<td>Gly</td><td rowspan="2">Gly</td><td>Thr</td><td>ala</td><td>ala</td><td>Leu</td><td rowspan="2">Gly</td><td>cys</td><td>Leu</td><td>val</td><td>lys</td><td>ASP</td><td rowspan="2">Tyr</td><td>phe</td><td>Pro</td><td>Glu</td>

<td>145</td><td></td><td></td><td></td><td>150</td><td></td><td></td><td></td><td>155</td><td></td><td></td><td></td><td>160</td>

<td>Pro</td><td>val</td><td>Thr</td><td>val</td><td>Cheese</td><td rowspan="2">Trp</td><td>own</td><td>Cheese</td><td rowspan="2">Gly</td><td>ala</td><td>Leu</td><td>Thr</td><td>Cheese</td><td rowspan="2">Gly</td><td>val</td><td>His</td>

<td></td><td></td><td></td><td></td><td>165</td><td></td><td></td><td>170</td><td></td><td></td><td></td><td>175</td><td></td>

<td>Thr</td><td>phe</td><td>Pro</td><td>ala</td><td>val</td><td>Leu</td><td>Gin</td><td>Cheese</td><td>Cheese</td><td rowspan="2">Gly</td><td>Leu</td><td rowspan="2">Tyr</td><td>Cheese</td><td>Leu</td><td>Cheese</td><td>Cheese</td>

<td></td><td></td><td></td><td>180</td><td></td><td></td><td></td><td></td><td>185</td><td></td><td></td><td>190</td><td></td><td></td>

<td>val</td><td>val</td><td>Thr</td><td>val</td><td>Pro</td><td>Cheese</td><td>Cheese</td><td>Cheese</td><td>Leu</td><td rowspan="2">Gly</td><td>Thr</td><td>Gin</td><td>Thr</td><td rowspan="2">Tyr</td><td>how much</td><td rowspan="2">Cys</td>

<td></td><td></td><td>195</td><td></td><td></td><td></td><td></td><td>200</td><td></td><td></td><td></td><td>205</td><td></td>

<td>own</td><td>val</td><td>own</td><td>His</td><td rowspan="2">lys</td><td>Pro</td><td>Cheese</td><td>own</td><td>Thr</td><td rowspan="2">lys</td><td>val</td><td>Asp</td><td></td><td></td><td></td><td></td>

<td></td><td>210</td><td></td><td></td><td></td><td>215</td><td></td><td></td><td></td><td>220</td><td></td><td></td><td></td><td></td>

&lt;210&gt; 17 &lt;211&gt; 660 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; oligonucleotide primer &lt;400&gt; 17 caggtacagc tggaggagtc agggggaggc gtggtccagc ctgggaggtc cctcagactc 60 tcctgtgcag cgtctggatt caccttcact aattatggca tgcactgggt ccgccaggct 120 ccaggcaagg gactcgagtg gctggcactc atatcctatg atggaaatag gcaatactat 180 gcagactccg tgaagggccg attcaccgtc tccagagaca atcccaacaa cacactgcat 240 ctggagatga agagcctgcg agccgaagac tcggctatat attactgtgc gagaggggct 300 ggggtattac tgtggttcgg cgacttatcc tggttcgacc cctggggcca gggaaccctg 360 gtcaccgtct cctcagcctc caccaagggc ccatcggtct tccccctggc accctcctcc 420 aagagcacct ctgggggcac agcggccctg ggctgcctgg tcaaggacta cttccccgaa 480 ccggtgacgg tgtcgtggaa ctcaggcgcc ctgaccagcg gcgtgcacac cttcccggct 540 gtcctacagt cctcaggact ctactccctc agcagcgtgg tgaccgtgcc ctccagcagc 600 ttgggcaccc agacctacat ctgcaacgtg aatcacaagc ccagcaacac caaggtggac 660 &lt;210&gt; 18 &lt;211&gt; 214 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic build &lt;400&gt; 18

<td colspan="5">asd Ile Gln Met Thr</td><td rowspan="2">Gin</td><td rowspan="2">Cheese</td><td colspan="8">Pro Ser Asn Leu Ser Ala Ser Val</td><td rowspan="2">Gly</td>

<td>1</td><td colspan="4">5</td><td colspan="6">10</td><td colspan="2">15</td>

<td rowspan="2">Asp</td><td rowspan="2">Arg</td><td>val</td><td>Thr</td><td>how much</td><td>Thr</td><td>Cys</td><td>Arg</td><td>ala</td><td>Cheese</td><td>Gin</td><td>own</td><td>how much</td><td>own</td><td>Thr</td><td>Trp</td>

<td></td><td>20</td><td></td><td></td><td></td><td></td><td>25</td><td></td><td></td><td></td><td></td><td>thirty</td><td></td><td></td>

<td>Leu</td><td>ala</td><td>Trp</td><td rowspan="2">Tyr</td><td>Gin</td><td>His</td><td>lys</td><td>pro</td><td>Gly</td><td>lys</td><td>Pro</td><td>Pro</td><td>lys</td><td>Leu</td><td>Arg</td><td>how much</td>

<td></td><td></td><td>35</td><td></td><td></td><td></td><td>40</td><td></td><td></td><td></td><td></td><td>45</td><td></td><td></td><td></td>

<td rowspan="2">Tyr</td><td>Gin</td><td>ala</td><td>Cheese</td><td>Thr</td><td>Leu</td><td>Glu</td><td>Cheese</td><td rowspan="2">Gly</td><td>val</td><td>Pro</td><td>Cheese</td><td>Arg</td><td>phe</td><td>Cheese</td><td>Gly</td>

<td>50</td><td></td><td></td><td></td><td></td><td>55</td><td></td><td></td><td></td><td>60</td><td></td><td></td><td></td><td></td>

<td>Cheese</td><td rowspan="2">Gl</td><td>Cheese</td><td rowspan="2">Gly</td><td>Thr</td><td>How much</td><td>phe</td><td>Thr</td><td>Leu</td><td>Thr</td><td>How much</td><td>Cheese</td><td>Cheese</td><td>Leu</td><td>Gin</td><td>Pro</td>

<td>65</td><td></td><td></td><td>70</td><td></td><td></td><td></td><td></td><td>75</td><td></td><td></td><td></td><td></td><td>80</td>

<td>Asp</td><td>Asp</td><td>phe</td><td>Gly</td><td>Thr 85</td><td>Tyr</td><td>Tyr</td><td>Cys</td><td>Gin</td><td>Gin 90</td><td>own</td><td>own</td><td>Cheese</td><td>Tyr</td><td>Pro 95</td><td>Tyr</td>

<td>Thr</td><td>phe</td><td rowspan="2">Gly</td><td>Gin</td><td rowspan="2">Gly</td><td>Thr</td><td rowspan="2">lys</td><td>Leu</td><td>Glu</td><td>how much</td><td>own</td><td rowspan="2">Arg</td><td>Thr</td><td>val</td><td>ala</td><td>ala</td>

<td></td><td></td><td>100</td><td></td><td></td><td>105</td><td></td><td></td><td></td><td>110</td><td></td><td></td>

<td>Pro</td><td>Cheese</td><td>val</td><td>phe</td><td>How much</td><td>phe</td><td>Pro</td><td>Pro</td><td>Cheese</td><td rowspan="2">Asp</td><td>Glu</td><td>Gin</td><td>Leu</td><td rowspan="2">lys</td><td>Cheese</td><td>Gly</td>

<td></td><td></td><td>115</td><td></td><td></td><td></td><td></td><td>120</td><td></td><td></td><td></td><td>125</td><td></td><td></td>

<td>Thr</td><td>ala</td><td>Cheese</td><td>val</td><td>val</td><td rowspan="2">Cys</td><td>Leu</td><td>Leu</td><td>own</td><td>own</td><td>phe</td><td>Tyr</td><td>Pro</td><td rowspan="2">Arg</td><td>Glu</td><td>ala</td>

<td></td><td>130</td><td></td><td></td><td></td><td>135</td><td></td><td></td><td></td><td></td><td>140</td><td></td><td></td><td></td>

<td>lys</td><td>val</td><td>Gin</td><td rowspan="2">Trp</td><td rowspan="2">lys</td><td>val</td><td>ASP</td><td>own</td><td>ala</td><td>Leu</td><td>Gin</td><td>Cheese</td><td>Gly</td><td>own</td><td>Cheese</td><td>Gin</td>

<td>145</td><td></td><td></td><td>150</td><td></td><td></td><td></td><td></td><td>155</td><td></td><td></td><td></td><td></td><td>160</td>

<td>Glu</td><td>Cheese</td><td>val</td><td>Thr</td><td>Glu</td><td>Gin</td><td rowspan="2">ASP</td><td>Cheese</td><td>Underworld</td><td>Asp</td><td>Cheese</td><td>Thr</td><td rowspan="2">Tyr</td><td>Cheese</td><td>Leu</td><td>Cheese</td>

<td></td><td></td><td></td><td></td><td>165</td><td></td><td></td><td></td><td>170</td><td></td><td></td><td></td><td>175</td><td></td>

<td>Cheese</td><td>Thr</td><td>Leu</td><td>Thr</td><td>Leu</td><td>Cheese</td><td rowspan="2">lys</td><td>ala</td><td>Asp</td><td>Tyr</td><td>Glu</td><td>lys</td><td>Hi s</td><td>lys</td><td>val</td><td>Tyr</td>

<td></td><td></td><td></td><td>180</td><td></td><td></td><td></td><td>185</td><td></td><td></td><td></td><td></td><td>190</td><td></td><td></td>

<td>ala</td><td>Cys</td><td>Glu</td><td>val</td><td>Thr</td><td>His</td><td>Gin</td><td>Gly</td><td>Leu</td><td>Cheese</td><td>Cheese</td><td>Pro</td><td>val</td><td>Thr</td><td>lys</td><td>Cheese</td>

195,200 205

Phe Asn Arg Gly Glu Cys 210 &lt;210&gt; 19 &lt;211&gt; 645 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; oligonucleotide primer &lt;400&gt; 19 gatatccaga tgacccagtc tccttccaac ctgtctgcat ctgtaggaga cagagtcaca atcacttgtc gggccagtca aaatattaat acctggctgg cctggtatca gcacaaacca gggaaacccc ctaagctccg gatatatcag gcgtctacgt tagaaagtgg ggtcccttca aggttcagcg gcagtggatc tgggacgata ttcactctca ccatcagcag cctgcagcct gatgattttg gaacttatta ctgccaacag aataatagtt acccgtacac ttttggccag gggaccaagc tggagatcaa ccgaactgtg gctgcaccat ctgtcttcat cttcccgcca tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag gagagtgtca cagagcagga cagcatggac agcacctaca gcctcagcag caccctgacg ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt GTTA &lt;210&gt; 20 &lt;211&gt; 20 &lt;212&gt; DNA &lt;213&gt; artificial sequence &lt;220&gt;

&lt;223&gt; An oligonucleotide primer &lt;400&gt; 20 tcaccatcca ttgcacagtt 20 &lt;210&gt; 21 &lt;211&gt; 20 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; An oligonucleotide primary &lt;400&gt; 21 ctgcgagaag gtactcaccc 20 &lt;210&gt; 22 &lt;211&gt; 23 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic build &lt;400&gt; 22

Gly Ala His Arg Thr Tyr Tro Gly His Thr Gly Ala Cys Asx Cys 15 10 15

Ala Gly Thr Cys Thr Cys cys 20 &lt;210&gt; 23 &lt;211&gt; 58 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; An oligonucleotide primer &lt;400&gt; 23 gatcgaattc ttaacactct cccctgttga agctctttgt gacgggcgag ctcaggcc &lt;210&gt; 24 &lt;211&gt; 21 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic build &lt;400&gt; 24

Ala Gly Gly Thr Arg Cys Ala Gly Cys Thr Gly Asx Trp Gly Ala '15 10 15

Gly Thr Cys Asp Gly 20 &lt;210&gt; 25 &lt;211&gt; 24 &lt;212&gt; DNA &lt;213&gt; Artificial Sequence &lt;220&gt;

&lt;223&gt; An oligonucleotide primary &lt;400&gt; 25 gtccacctgg gtgttgctgg gctt 24 &lt;210&gt; 26 &lt;211&gt; 23 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; An oligonucleotide primary &lt;400&gt; 26 agcggataac aatttcacac agg &lt;210&gt; 27 &lt;211&gt; 24 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; An oligonucleotide primary &lt;400&gt; 27 cgccagggtt ttcccagtca cgac 24 &lt;210&gt; 28 &lt;211&gt; 99 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Oligonucleotide primer &lt;400&gt; 28 gatcggatcc gccgccacca tgggatggag tacaggtgta cacagcgaaa ttgtgctgac ctcttcttgg tagcaacagc tcagtctcc &lt;210&gt; 29 &lt;211&gt; 35 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; An oligonucleotide primer &lt;400&gt; 29 gatcgaattc tcatttcccg ggagacaggg agagg 35 &lt;210&gt; 30 &lt;211&gt; 105 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Oligonucleotide primer &lt;400&gt; 30 gatcggatcc aagcttgccg ccaccatggg atggagctgt atcatcctct tcttggagag 60 a gmggtacca ggggtgca gctggtggag tctgg 105 &lt;210&gt; 31 &lt;211&gt; 20 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; An oligonucleotide primer &lt;400&gt; 31 ggagaagaaa gttgagccca 20 &lt;210&gt; 32 &lt;211&gt; 20 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; An oligonucleotide primer &lt;400&gt; 32 tgcaaggtct ccaacaaagc 20 &lt;210&gt; 33 &lt;211&gt; 20 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; An oligonucleotide primer &lt;400&gt; 33 cctggttctt ggtcagctca 20 &lt;210&gt; 34 &lt;211&gt; 20 &lt;212&gt; DNA &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; An oligonucleotide primary &lt;400&gt; 34 ggcacggtgg ggatgtgtga 20 &lt;210&gt; 35 &lt;211&gt; 12 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic build &lt;400&gt; 35

Ala Arg Arg Leu Leu Asn Leu cheese Arg Asp Thr Ala 1 5 10 &lt;210&gt; 36 &lt;211&gt; 12 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic build &lt;400&gt; 36

Thr Arg Leu Glu Leu Tyr Lys Gln Gly Leu Arg Gly 1 5 10 &lt;210&gt; 37 &lt;211&gt; 12 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic build &lt;400&gt; 37

Tyr Lys Gln Gly Leu Arg Gly cheese Leu Thr Lys Leu 15 10 &lt;210&gt; 38 &lt;211&gt; 12 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic build &lt;400&gt; 38

Arg Gly Leu Thr Leys Leu Lys Gly Pro Leu Thr 15 10 &lt;210&gt; 39 &lt;211&gt; 12 &lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt;

&lt;223&gt; Synthetic build &lt;400&gt; 39

Lys Glu Asn Leu Lys Asp Phe Leu Val How much Pro 1 5 10 &lt;210&gt; 40 &lt;211&gt; 10 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic build &lt;400&gt; 40

Gly Phe Thr Phe Ser Arg His Trp Met His 1 5 10 &lt;210&gt; 41 &lt;211&gt; 17 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic build &lt;400&gt; 41

Search Ile Asn Gly Ala Gly Thr Ser Ile Thr Ala Asp Ser Val Arg 1 5 10 15

Gly &lt;210&gt; 42 &lt;211&gt; 12 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic build &lt;400&gt; 42

Ala Asn Ser val Trp Phe Arg Gly Leu Phe Asp Tyr 1 5 10 &lt;210&gt; 43 &lt;211&gt; 11 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic build &lt;400&gt; 43

Arg Ala Ser Gln Series Val Thr Asn Leu Ala 1 5 10 &lt;210&gt; 44 &lt;211&gt; 7 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic build &lt;400&gt; 44

Gly Ala Ser Thr Arg Ala Thr &lt;210&gt; 45 &lt;211&gt; 9 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic build &lt;400&gt; 45

Gln Gln Tyr Asp Lys Trp Pro Asp Thr &lt;210&gt; 46 &lt;211&gt; 10 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic build &lt;400&gt; 46

Gly Phe Thr Phe Thr Asn Tyr Gly Met His 1 5 10 &lt;210&gt; 47 &lt;211&gt; 17 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic build &lt;400&gt; 47

Leu Ile Ser Tyr Asp Gly Asn Arg Gln Tyr Tyr Ala Asp Ser val Lys 15 10 15

Gly &lt;210&gt; 48 &lt;211&gt; 16 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic build &lt;400&gt; 48

Gly Ala Gly Val Leu Tru Phe Gly Asp Leu Ser Trp Phe Asp Pro 15 10 15 &lt;210&gt; 49 &lt;211&gt; 11 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic build &lt;400&gt; 49

Arg Ala Ser Gln Asn Asn Thr Trp Leu Ala 1 5 10 &lt;210&gt; 50 &lt;211&gt; 7 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic build &lt;400&gt; 50

Gln Ala Ser Thr Leu Glu Ser 1 5 &lt;210&gt; 51 &lt;211&gt; 9 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic build &lt;400&gt; 51

Gln Gln Asn Asn Cheese Tyr Pro Tyr Thr 1 5 &lt;210&gt; 52 &lt;211&gt; 25 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic build &lt;400&gt; 52

Glu val Gln Leu val Glu Cheese Gly Gy Gy Leu val Gn Pro Gly Gly 15 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser 20 25 &lt;210&gt; 53 &lt;211&gt; 14 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic build &lt;400&gt; 53

Trp Leu Arg Gln Val Pro Gy Lys Gy Pro val Trp val Ser 15 10 &lt;210&gt; 54 &lt;211&gt; 32 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic build &lt;400&gt; 54

Arg Phe Thr How much Arg Asp Asa Asn Asn Thr Leu Phe Leu Gln 15 10 15

Met Asn Leu Ser Arg Ala Asp Asp Thr Ala Leu Ti Phe Ce Ala Arg 20 25 30 &lt;210&gt; 55 &lt;211&gt; 23 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic build &lt;400&gt; 55

Glu Ile Val Leu Thr Gln Cheese Pro Thr Leu Cheese val Cheese Pro Gly 15 10 15

Glu Arg val Thr Leu cheese Cys 20 &lt;210&gt; 56 &lt;211&gt; 15 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic build &lt;400&gt; 56

Trp Tyr Gln Gln Lys Leu Gly Gln Gly Pro LeLu Leu Ile Tyr 15 10 15 &lt;210&gt; 57 &lt;211&gt; 32 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic build &lt;400&gt; 57

Asp Ile Pro Ala Arg Phe Cheese Gly Cheese Gly Cheese Glu Thr Glu Phe Thr 15 10 10

Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp Phe Ala Val Tyr Tyr Cys 20 25 30 &lt;210&gt; 58 &lt;211&gt; 25 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic build &lt;400&gt; 58

Gln val Gln Leu Glu Glu Cheese Gly Gly Gly Val Val Gln Pro Gly Arg 15 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser 20 25 &lt;210&gt; 59 &lt;211&gt; 14 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic build &lt;400&gt; 59

Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Leu Ala 1 5 10 &lt;210&gt; 60 &lt;211&gt; 32 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic build &lt;400&gt; 60

<td>Arg 1</td><td>phe</td><td>Thr</td><td>val</td><td>Cheese 5</td><td>Arg</td><td colspan="7">Asp Asn Asn Asn Thr Leu 10</td><td>His</td><td>Leu 15</td><td>Glu</td>

<td>Underworld</td><td rowspan="2">lys</td><td>Cheese</td><td>Leu</td><td rowspan="2">Arg</td><td>ala</td><td>Glu</td><td rowspan="2">Asp</td><td>Cheese</td><td>ala</td><td>how much</td><td>Tyr</td><td>Tyr</td><td>Cys</td><td>ala</td><td>Arg</td>

<td></td><td></td><td>20</td><td></td><td></td><td>25</td><td></td><td></td><td></td><td></td><td>thirty</td><td></td><td></td>

&lt;210&gt; 61 &lt;211&gt; 23 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic build &lt;400&gt; 61

Asp Ile Gln Met Thr Gln Cheese Pro Cheese Asn Leu Cheese Ala Cheese Val Gly

10 15

Asp Arg Val Thr Ile Thr Cys 20 &lt;210&gt; 62 &lt;211&gt; 15 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic build &lt;400&gt; 62

Trp Tyr Gn His Lys Pro Gy Lys Pro Pro Lys Leu Arg Ile Tyr 15 10 15 &lt;210&gt; 63 &lt;211&gt; 32 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic build

<td>&lt;400&gt; 63</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>Gly</td><td>val</td><td>Pro</td><td>Cheese</td><td>Arg</td><td>phe</td><td>Cheese</td><td rowspan="2">Gly</td><td>Cheese</td><td>Gly</td><td>Cheese</td><td rowspan="2">Gly</td><td>Thr</td><td>How much</td><td>Phe Thr</td>

<td>1</td><td></td><td></td><td></td><td>5</td><td></td><td></td><td></td><td>10</td><td></td><td></td><td></td><td>15</td>

<td>Leu</td><td>Thr</td><td>How much</td><td>Cheese</td><td>cheese</td><td>Leu</td><td>Gln</td><td>Pro</td><td>Asp</td><td rowspan="2">Asp</td><td>phe</td><td rowspan="2">Gly</td><td>Thr</td><td>Tyr</td><td rowspan="2">Tyr Cys</td>

<td></td><td></td><td></td><td>20</td><td></td><td></td><td></td><td></td><td>25</td><td></td><td></td><td>thirty</td>

&lt;210&gt; 64 &lt;211&gt; 82 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic build &lt;400&gt; 64

<td colspan="5">Cys Ser Ile Ser Ala</td><td colspan="2" rowspan="2">Pro Al a</td><td rowspan="2">Arg</td><td colspan="8">Pro cheese Pro Pro cheese Thr Gln Pro</td>

<td colspan="3">1</td><td colspan="2">5</td><td colspan="2">10</td><td colspan="6">15</td>

<td rowspan="2">Trp</td><td>Glu</td><td>His</td><td>val</td><td>own</td><td>ala</td><td>How much</td><td>Gln</td><td>Glu</td><td>ala</td><td rowspan="2">Arg</td><td rowspan="2">Arg</td><td>Leu</td><td>Leu</td><td>own</td><td>Leu</td>

<td></td><td></td><td>20</td><td></td><td></td><td></td><td></td><td>25</td><td></td><td></td><td>thirty</td><td></td><td></td>

<td>Cheese</td><td rowspan="2">Arg</td><td>Asp</td><td>Thr</td><td>ala</td><td>ala</td><td>Glu</td><td>Underworld</td><td>own</td><td>Glu</td><td>Thr</td><td>val</td><td>Glu</td><td>val</td><td>how much</td><td>Cheese</td>

<td></td><td>35</td><td></td><td></td><td></td><td></td><td>40</td><td></td><td></td><td></td><td></td><td>45</td><td></td><td></td><td></td>

<td>Glu</td><td>Underworld</td><td>phe</td><td rowspan="2">Asp</td><td>Leu</td><td>Gin</td><td>Glu</td><td>pro</td><td>Thr</td><td rowspan="2">cys</td><td>Leu</td><td>Gl n</td><td>Thr</td><td rowspan="2">Arg</td><td>Leu</td><td>Glu</td>

<td></td><td>50</td><td></td><td></td><td></td><td>55</td><td></td><td></td><td></td><td>60</td><td></td><td></td><td></td>

<td colspan="5">Leu Tyr Lys Gin Gly</td><td>Lei</td><td colspan="10">and Arg Gly Leu Thr Leys Leu Lys Gly Pro</td>

<td>65</td><td></td><td></td><td></td><td></td><td>70</td><td></td><td></td><td></td><td></td><td>75</td><td></td><td></td><td></td><td></td><td>80</td>

Leu Thr &lt;210&gt; 65 &lt;211&gt; 12 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic build &lt;400&gt; 65 cys ser How How Ser Ala Pro Ala Arg cheese Pro Ser Pro 1 5 10 &lt;210&gt; 66 &lt;211&gt; 12 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic build &lt;400&gt; 66

Pro Ala Arg Ser Pro Ser Pro cheese Thr Gln pro Trp 1 5 10 &lt;210&gt; 67 &lt;211&gt; 12 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic build &lt;400&gt; 67

Ser pro Ser Thr Gln Pro Trp Glu His val Asn Ala 1 5 10 &lt;210&gt; 68 &lt;211&gt; 12 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic build &lt;400&gt; 68

Pro Trp Glu His Val Asn Ala Ile Gln Glu Ala Arg

5 10 &lt;210&gt; 69 &lt;211&gt; 12 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic build &lt;400&gt; 69

Asn Ala ne kadar Gin Glu Alu Arg Arg Leu Leu Asn Leu 1 5 10 &lt;210&gt; 70 &lt;211&gt; 12 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic build &lt;400&gt; 70

Asn Leu Ser Arg Asp Thr Ala Ala Glu Met Asn Glu 1 5 10 &lt;210&gt; 71 &lt;211&gt; 12 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic build &lt;400&gt; 71

Thr Ala Ala Glu Met Asn Glu Thr Val Glu val as 1 5 10 &lt;210&gt; 72 &lt;211&gt; 12 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic build &lt;400&gt; 72

Asn Glu Thr val Glu Val Ile Ser Glu Met Phe Asp 1 5 10 &lt;210&gt; 73 &lt;211&gt; 12 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic build &lt;400&gt; 73 Ile Ser Glu Met Phe Asp Leu Gin Glu pro Thr 15 10 &lt;210&gt; 74 &lt;211&gt; 12 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic build &lt;400&gt; 74

Phe asd Leu Gln Glu Pro Thr Cys Leu Gln Thr Arg 15 10 &lt;210&gt; 75 &lt;211&gt; 12 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic build &lt;400&gt; 75

Pro Thr Cys Leu Gln Thr Arg Leu Glu Leu Tyr Lys 1 5 10 &lt;210&gt; 76 &lt;211&gt; 56 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic build &lt;400&gt; 76

<td>lys</td><td>Leu</td><td>lys</td><td>Gly</td><td>pro 5</td><td>Leu</td><td>Thr</td><td>Underworld</td><td>Underworld</td><td>ala 10</td><td>Cheese</td><td>Hi s</td><td>Tyr</td><td>lys</td><td>Gln 15</td><td>His</td>

<td>cys</td><td>Pro</td><td>Pro</td><td>Thr 20</td><td>pro</td><td>Glu</td><td>Thr</td><td>Cheese</td><td>cys 25</td><td>ala</td><td>Thr</td><td>Gln</td><td>how much</td><td>how much thirty</td><td>Thr</td><td>phe</td>

<td>Glu</td><td>Cheese</td><td>phe 35</td><td>lys</td><td>Glu</td><td>own</td><td>Leu</td><td>lys 40</td><td>Asp</td><td>phe</td><td>Leu</td><td>Leu</td><td>val 45</td><td>how much</td><td>Pro</td><td>phe</td>

<td>Asp</td><td>cys 50</td><td>Trp</td><td>Glu</td><td>Pro</td><td>val</td><td>Gln 55</td><td>Glu</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

&lt;210&gt; 77 &lt;211&gt; 12 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic build &lt;400&gt; 77

Lys Leu Lys Gly Pro Leu Thr Met Met Ala Ser His 15 10 &lt;210&gt; 78 &lt;211&gt; 12 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic build &lt;400&gt; 78

Leu Thr Met Met Ala Ser His Tyr Lys Gln His Cys 1 5 10 &lt;210&gt; 79 &lt;211&gt; 12 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic build &lt;400&gt; 79

Ser His Tyr Lys Gln His Cys Pro Pro Thr Pro Glu 1 5 10 &lt;210&gt; 80 &lt;211&gt; 12 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic build &lt;400&gt; 80

His Cys Pro Pro Thr Pro Glu Thr Ser Cys Ala Thr 1 5 10 &lt;210&gt; 81 &lt;211&gt; 12 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic build &lt;400&gt; 81 pro Glu Thr Ser Cys Ala Thr Gln How much Thr Phe 1 5 10 &lt;210&gt; 82 &lt;211&gt; 12 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic build &lt;400&gt; 82

Ala Thr Gln how much Thr Phe Glu cheese Phe Lys Glu 15 &lt;210&gt; 83 &lt;211&gt; 12 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic build &lt;400&gt; 83

Thr Phe Glu cheese Phe Lys Glu Asn Leu Lys Asp Phe 15 10 &lt;210&gt; 84 &lt;211&gt; 12 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic build &lt;400&gt; 84

Asp Phe Leu Leu val How much Pro Phe Asp Cys Trp Glu 15 10 &lt;210&gt; 85 &lt;211&gt; 11 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic build as Pro Phe Asp Cys Trp Glu Pro val Gln Glu 1 5 10 &lt;400&gt; 85 &lt;210&gt; 86 &lt;211&gt; 12 &lt;212&gt; PRT &lt;213&gt; Artificial sequence &lt;220&gt;

&lt;223&gt; Synthetic build &lt;400&gt; 86

Gly Glu Gln Lys Leu How much Glu Glu Asp Cys Gly 1 5 10