MXPA06008690A - IDENTIFICATION OF NOVEL IgE EPITOPES - Google Patents

Abstract

The present invention relates to novel peptide epitopes derived from the CH3 domain of IgE which are recognized by high affinity antibodies that specifically bind IgE. These novel peptides may be used for both active immunization of a subject by administering these peptides to generate high affinity antibodies in a subject, as well as for generating high affinity anti-IgE antibodies in non-human hosts that specifically bind to these regions of IgE for passive immunization of a subject.

Description

IDENTIFICATION OF EPITOPOS gE NOVEDOSOS CROSS REFERENCE WITH RELATED APPLICATIONS This application claims the priority of PCT applications No. PCT / US04 / 02892 and PCT / US04 / 02894 filed on February 2, 2004, both are incorporated by reference to this application.

BACKGROUND OF THE INVENTION Allergy is a state of hypersensitivity induced by an exaggerated immune response to a foreign agent, such as, for example, an allergen. Immediate hypersensitivity (type I), characterized by allergic reactions immediately after contact with an allergen, is mediated via B cells and is based on antigen-antibody reactions. Delayed hypersensitivity is mediated via T cells and is based on mechanisms of cellular immunity. In recent years, the term "allergy" has become increasingly synonymous with type I hypersensitivity. Immediate hypersensitivity is a response based on the production of antibodies to class E immunoglobulin (IgE antibodies) by B cells that, with exposure to a differentiated allergen in an antibody, they secrete plasma cells. The IgE-induced reaction is a local event that occurs at the site of the entry of the allergen into the body, that is, into mucosal surfaces and / or lymph nodes. The first locally produced IgE will be sensitive to local mast cells, that is, to IgE antibodies bound with their constant regions to the Fce receptors on the surface of mast cells, and subsequently the "excess" IgE enters the circulation and binds to the receptors in the circulating basophils as well as in the mast cells fixed in the tissue throughout the body. When the linked IgE subsequently makes contact with the allergen, the Fce receptors are cross-linked with the allergen binding, which causes the cells to degrade and releases a variety of anaphylactic mediators, such as histamine, prostaglandins, leukotrienes, etc. . It is the release of these substances that is responsible for the typical clinical symptoms of immediate hypersensitivity, particularly the contraction of smooth muscle in the respiratory tract or in the intestine, the dilation of small blood vessels and the increase in their water permeability and to plasma proteins, the resulting mucous secretion, for example, allergic rhinitis, eczema and atopic asthma, and the stimulation of the nerve terminals in the skin which produces jaundice and pain. In addition, the reaction after the second contact with the allergen is intensified because some B cells form a "memory segment" of surface IgE positive B cells (sIgE + B cells) after the first contact with the allergen when expressing IgE on the cell surface. There are two main IgE receptors, the high affinity FceRI receptor, and the low affinity FceRII receptor. The FceRI receptor is predominantly expressed on the surface of mast cells and basophils, but low levels of the FceRI receptor can also be found in Langerhan cells, dendritic cells and monocytes, where it functions in mediated allergen presentation. In addition, it has been reported in human platelets and eosinophils (Hasega a, S. et al., Hematopoiesis, 1999, 93: 2543-2551). The FceRI receptor was not found on the surface of B cells, T cells or neutrophils. The expression of the FceRI receptor in Langerhan cells and dermal dendritic cells is functionally and biologically important for the presentation of the IgE-linked antigen in allergic individuals (Klubal R. et al., J. Invest, Dermatol, 1997, 108 (3): 336-42). The low affinity receptor, FceRII (CD23) is a lectin-like molecule that comprises three identical subunits with major structures that extend from a long coiled stem (stem) to helical of the cellular plasma membrane (Dierks, AE et al. ., 1993, 150: 2372-2382). When linking to Ige; the FceRII receptor associates with CD21 in the B cells involved in the regulation of IgE synthesis (Sanon, A. et al., J. Allergy Clin, Immunol., 1990, 86: 333-344, Bonnefoy, J. et al. ., Eur. Resp. J. 1996, 9: 63s-66s). The FceRII receptor has been widely recognized for allergen presentation (Sutton and Gould, 1993, Nature, 366: 421-428). IgE bound to FceRII in epithelial cells is responsible for the rapid and specific presentation of allergen (Yang, P.P. J. Clin.Invest., 2000, 106: 879-886). The FceRII receptor occurs in various cell types, including B cells, eosinophils, platelets, natural killer cells, follicular dendritic cells and Langerhan cells. The structural entities in the IgE molecule that interact with FceRI and FceRII have also been identified. Mutagenesis studies have indicated that the CH3 domain mediates the interaction of IgE with both the FceRI receptor (Presta et al., J. Biol. Chem. 1994, 269: 26368-26373, Henry AJ et al., Biochemistry, 1997, 36 : 15568-15578) as well as with the FceRII receptor (Sutton and Gould, Na ture, 1993, 366: 421-428; Shi, J. et al., Biochemistry, 1997, 36: 2112-2122). The binding sites for the high and low affinity receptors are located symmetrically along a central rotational axis through the two CH3 domains. The FceRI receptor binding site is located in a CH3 domain on the outer side near the binding of the CH2 domain, whereas the FceRII receptor binding site is located at the carboxyl-terminus of CH3. A promising concept for the treatment of allergy involves the application of monoclonal antibodies, which are specific to the IgE isotype and thereby are capable of binding to IgE. This methodology is based on the inhibition of allergic reactions by down-regulating the immune response of IgE, which is the first event in the induction of allergy and provides maintenance of the allergic state. Since the immune response to the response of other antibody classes, an immediate effect as well as a lasting effect on allergic symptoms is achieved. The first density studies of human basophils showed a correlation between the level of IgE in the plasma of a patient and the number of FceRI receptors per basophil (Malveaux et al., J. Clin. Invest., 1978, 62: 176). These studies did not note that FceRI densities in allergic and non-allergic people range from 104 to 106 recipients per basophil. It was later shown that the treatment of allergic diseases with anti-IgE decreases the amount of circulating IgE to 1% of the levels prior to treatment (MacGlashan et al., J. Immunol., 1997, 158: 1438-1445). MacGlashan analyzed serum obtained from patients treated with complete anti-IgE antibody, which binds to free IgE circulating in the patient's serum. He and his collaborators reported that decreasing the level of circulating IgE in a patient produces a lower number of receptors present on the surface of basophils. Therefore, they hypothesized that the density of FceRI on the surface of basophils and mast cells is directly or indirectly regulated by the level of the circulating IgE antibody. More recently, WO 99/62550 described the use of IgE molecules and fragments, which bind to the IgE binding sites of FceRI and FceRII. However, effective therapies that have no side effects from the management of these allergic diseases are limited. A therapeutic methodology for treating allergic diseases involved using humanized anti-IgE antibody to treat asthma and allergic rhinitis (Corne, J. et al., J. Clin. Invest. 1991, 99: 879-887; Racine-Poon, A. et al., Clin. Pharmcol., Ther. 1997, 62: 675-690, Fahy, JVJ et al., Am. J. Resp. Crit. Care Med. 1997, 155: 1824-1834; Boulet, LP et al. , Am. J. Resp. Crit. Care Med. 1997, 155: 1835-1840; Milgrom, E. et al., N. Engl. J. Med., 1999, 341: 1966-1973). These clinical data show that the inhibition of IgE binding to its receptors is an effective methodology for treating allergic diseases. Antibodies suitable as anti-allergic agents will react with B cells positive for surface IgE, which differentiate in the plasma cells that produce IgE, so that these antibodies can be used to functionally remove these B cells. However, Antibodies to IgE in principle can also induce mediating release from mast cells sensitized to IgE by crosslinking the Fce receptors, thereby antagonizing the beneficial effect exerted at the serum level of the cells and IgE. One of the potentially dangerous problems in the development of anti-IgE therapies is the possibility of cross-linking IgE caused by the binding of therapeutic antibody to IgE already bound to the high affinity receptor and activating the release of histamine which produces a potentially reactive reaction. anaphylactic Therefore, antibodies applicable for allergy therapy should not react with IgE bound in basophils and sensitized mast cells, but should maintain the ability to recognize sIgE + B cells. These antibodies specific to the IgE isotype have been described, for example, by Chang et al. (Biotechnology 8, 122-126 (1990)), in European Patent No. EP0407392, and in various United States patents, for example, U.S. Patent No. 5,449,760. The peptides used to generate anti-IgE antibodies also suffer from the potential danger of inducing anaphylactic antibodies. The generation of anti-IgE antibodies during active vaccination may be able to activate the release of histamine in the same way as anti-IgE antibodies administered passively, if antibodies, generated during immunization, bind to bound IgE to the high affinity IgE receptor or by other mechanisms. Accordingly, there is a need for higher affinity non-anaphylactic antibodies that specifically bind to IgE, but do not bind to IgE already bound to its high affinity receptor, - as well as the need for peptides for immunization. activates that does not induce anaphylactic antibodies. The inventors have identified the specific epitope of IgE which provides a high affinity binding of the antibodies without binding to IgE in mast cells or basophils. These specific epitopes can, in turn, generate specific peptides for active immunization to generate antibodies to IgE that only bind to the IgE region that binds to the receptor, which ensures that the antibodies do not cross-link to IgE already bound to the receptor and therefore that are not anaphylactic SUMMARY OF THE INVENTION The present invention relates to novel peptide epitopes derived from the CH3 domain of IgE. These peptide epitopes are recognized by high affinity antibodies that specifically bind to IgE. These novel peptides can be used for the active immunization of a mammal by administering these peptides to generate high affinity antibodies in the mammal. Peptide epitopes can also be used in the generation of high affinity anti-IgE antibodies in a non-human host that specifically binds to these IgE regions and the resulting antibodies can be used for passive immunization of a mammal. An immunogen (epitope A, Figure 11) of the present invention comprises the amino acid sequence: Asn Pro Arg Gly Val Ser Xaa Tyr Xaa Xaa Arg Xaa (SEQ ID NO: 72). An example of the A epitope is: Asn Pro Arg Gly Val Ser Wing Tyr Leu Ser Arg Pro (SEQ ID NO: 73). Another immunogen (epitope B, Figure 11) comprises the amino acid sequence: Leu Pro Arg Ala Leu Xaa Arg Ser Xaa (SEQ ID NO: 74) Examples of Epitope B include: Leu Pro Arg Ala Leu Met Arg Ser Thr (SEQ ID NO: 75) His Pro His Leu Pro Arg Ala Leu Met Arg Ser Thr (SEQ ID NO: 76) Leu Pro Arg Ala Leu Met Arg Ser Thr Thr Lys Thr (SEQ ID NO: 77). In either SEQ ID NO: 72 or SEQ ID NO: 74, Xaa can be any amino acid. These peptides can be included in a composition containing at least one of the peptides and a physiologically acceptable carrier, diluent, stabilizer or excipient, as well as an immunogenic carrier. The immunogenic carrier can be, for example, BSA, KLH, tetanus toxoid and diphtheria toxoid. The present invention also relates to polynucleotides that encode SEQ ID NOS. 72-77, vectors comprising these polynucleotides and cells harboring said vectors. The present invention also relates to antibodies that specifically bind to the epitope A and / or epitope B. The present invention is also directed to a method for making antibodies that specifically bind to the epitope A and / or epitope. B.

The present invention relates to the administration of peptides comprising SEQ ID NO: 72 and / or SEQ ID NO: 74 to an individual suffering from a disease or condition mediated by IgE. The present invention relates to the administration of high affinity antibodies generated using peptides comprising SEQ ID NO: 72 and / or SEQ ID NO: 74 to a mammal suffering from a disease or condition mediated by IgE. The high affinity antibody can be human, humanized or chimeric. The antibody can be polyclonal or monoclonal. These diseases or conditions mediated by IgE include, for example, asthma, atopic dermatitis, urticaria, allergic rhinitis and eczema.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a schematic representation of the phage vector in the cloning and screening of antibody. Figure 2 is a schematic representation of oligonucleotides useful in the generation of antibody variants. Figure 3A describes the comparison of the light chains of TES-C21 murine anti-IgE antibody and the combined human template of L16 and JK4. Figure 3B describes the comparison of the heavy chains of TES-C21 and the combined human template DP88 and JH4b. Figure 4 presents a table of the variants of framework residues having high affinity compared to the parent TES-C21. Figures 5A and 5B describe the ELISA titration curves for clones 4, 49, 72, 78 and 136, compared to the progenitor Fab of TES-C21 and with the negative control (5D12). Figure 6 describes an inhibition assay of clones 2C, 5A and 51, compared to the parent TES-C21 and with the negative control antibody. Figure 7A depicts sequences of clones that have a combination of beneficial mutations that result in an even greater af affinity to IgE. Figures 8A and 8B describe the framework sequences of the entire light chain variable region for clones 136, 1, 2, 4, 8, 13, 15, 21, 30, 31, 35, 43, 44, 53, 81 , 90 and 113. Figures 9A and 9B describe the heavy chain complete variable framework sequences for 35 clones. Figures 10A-F describe the complete heavy and light chain sequences for clones 136, 2C, 51, 5A, 2B and 1136-2C.

Figure 11 depicts the amino acid sequence of CH3 region of human IgE and highlights Epitope "A" and Epitope "B". Figure 12 describes the overlapping peptides used to identify Epitope B. Figure 13 describes the identification of important residues in the binding region of Epitope A. Figure 14 describes the identification of important residues in the binding region of Epitope B Figure 15 describes a Western blot analysis of MAb that binds to mutant peptides. Figure 16 depicts the generation of anti-IgE antibodies in a transgenic animal expressing human IgE.

DETAILED DESCRIPTION OF THE INVENTION Definitions: The terms employed through this application are defined with the ordinary and typical meaning for those skilled in the art. However, the Requesters wish that the following terms are determined for the particular definition, as defined below. The phrase "virtually identical", with respect to an antibody chain polypeptide sequence, can be defined as an antibody chain exhibiting at least 70% or 80%, or 90% or 95% sequence identity with respect to the reference polypeptide sequence. The term, with respect to a nucleic acid sequence, can be defined as a nucleotide sequence that exhibits at least about 85% or 90%, or 95% or 97% sequence identity with respect to the nucleic acid sequence reference. The term "identity" or "homology" should be interpreted to mean the percentage of amino acid residues in the candidate sequence that are identical with the residue of a corresponding sequence with which it is compared, after alignment of the sequences and introduction of the openings, if it is necessary to achieve the maximum percentage of identity for the entire sequence, and without considering any conservative substitution as part of the sequence identity. Neither extensions nor N- or C-terminal insertions should be interpreted as identity or reductive homology. The methods and computer programs for alignment are generally known in the art. The sequence identity can be ensured using a sequence analysis software. The term "antibody" is used in the broadest sense, and specifically covers monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and multispecific antibodies (eg, bispecific antibodies). Antibodies (Abs) and immunoglobulins (Igs) are glycoproteins that have the same structural characteristics. While antibodies exhibit binding specificity towards a specific target, immunoglobulins include both antibodies and antibody-like molecules lacking target specificity. Immunoglobulins and native antibodies are usually heterotetrameric glycoproteins of approximately 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains. Each light chain has a variable domain at one end (VL) and a constant domain at its other end. The term "high affinity" antibodies refers to those antibodies that have a binding affinity of at least 10"10, preferably 10-12. As used herein, the term "Anti-human IgG antibody" means an antibody that binds to human IgE, so that it inhibits or considerably reduces the binding of IgE to the high affinity receptor, FceRI. The term "variable", in the context of the variable domain of antibodies, refers to the fact that certain portions of the variable domains differ widely in sequence among antibodies, and are used in the binding and specificity of each particular antibody for its particular purpose. Nevertheless, the variability is not distributed uniformly across the variable domains of the antibodies. It is concentrated in three segments called complementarity determining regions (CDR, complement ity determining region) also known as hypervariable regions in the variable domains of both the light chain and the heavy chain. The much more conserved portions of the variable domains are called the framework (FR). Each of the variable domains of native light and heavy chains comprises four FR regions, mostly an adoption of a ß-leaf configuration, connected by three CDRs, which form loops that connect, and in some cases are part of , the structure of ß-sheet. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the target binding site of the antibodies (see Kabat et al.). As used in this description, the amino acid residue numbering of the immunoglobulin is carried out according to the amino acid residue numbering system of the immunoglobulin of Kabat et al., (Sequences of Proteins of Immunological Interest, National Institute of Health, Bethesda, Md. 1987), unless otherwise indicated. The term "antibody fragment" refers to a portion of a full-length antibody, broadly speaking, the variable or target binding region. Examples of antibody fragments include Fab, Fab ', F (ab') 2 and Fv fragments. The phrase "analog or functional fragment" of an antibody means a compound having qualitative biological activity in common with a full-length antibody. For example, an analog or functional fragment of an anti-IgE antibody is one that can bind to an IgE immunoglobulin in such a way that the ability of this molecule to have the ability to bind to the high receptor is prevented or greatly reduced. affinity, FceRI. As used in this description, the term "functional fragment", with respect to antibodies, refers to Fv, F (ab) and F (ab ') 2 fragments. An "Fv" fragment is the minimum antibody fragment that contains a complete binding and target recognition site. This region consists of a dimer of the variable domain of a light chain and a heavy chain in a close, non-covalent association (VH-VL dimer). It is in this configuration that the three CDRs of each variable domain interact to define a target binding site on the surface of the VH-VL dimer. Together, the six CDRs confer targeting specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three specific CDRs for a target) has the ability to recognize and bind to the target, albeit at a lower affinity than the entire binding site. The "single chain Fv" or "sFv" antibody fragments comprise the VH-VL dimers of an antibody, wherein these domains are present in a single polypeptide chain. In general terms, the Fv polypeptide further comprises a polypeptide linker between the VH-VL dimers which enables the sFv to form the desired structure for target binding. The Fab fragment contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. The Fab 'fragments differ from the Fab fragments by the addition of a few residues at the carboxyl terminus of the heavy chain domain CH1 including one or more cysteines from the antibody hinge region. F (ab ') fragments are produced with the cleavage of the bisulfide bond in the hinge cysteines of the pepsin digestion product F (ab') 2-Additional chemical pairings of antibody fragments are known to those of ordinary experience in The technique. The term "monoclonal antibody", as used in this description, refers to an antibody obtained from a population of practically homogeneous antibodies, ie, the individual antibodies comprising the population are identical, with the exception of possible mutations of natural origin that can be Present in smaller quantities. Monoclonal antibodies are highly specific, - they are directed towards a single target site. Furthermore, in contrast to conventional antibody preparations (polyclonal) that usually include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed towards a single determinant, a single determinant in the target. In addition to their specificity, monoclonal antibodies are advantageous in that they can be synthesized by means of the hybridoma culture, without contamination by other immunoglobulins. The "monoclonal" modifier indicates the character of the antibody when it is obtained from a practically homogeneous population of antibodies, and does not mean that antibody production is required by any particular method. For example, monoclonal antibodies for use with the present invention can be isolated from phage antibody libraries using commonly used techniques. The monoclonal parent antibodies which are used according to the present invention can be made with the first hybridoma method described by Kohier and Milstein, Nature 256, 495 (1975), or they can be made by means of recombinant methods. The "humanized" forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as, for example, Fv, Fab, Fab1, F (ab ') 2 or other sequences, which are bind to the target, antibodies) that contain the minimal sequence derived from the non-human immunoglobulin. In general, the humanized antibody will comprise substantially all of at least two typically variable domains, all or virtually all CDR regions corresponding to those of a non-human immunoglobulin and all or virtually all FR regions are those of a consensus sequence of human immunoglobulin. The humanized antibody can also comprise at least a portion of a constant region (Fc) of immunoglobulin, usually that of a chosen human immunoglobulin template. The terms "cell", "cell line" and "cell culture" include the progeny. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Variant progeny that have the same biological function or function are included, since they were screened from the originally transformed cell. The "host cells" employed in the present invention, in general terms, are prokaryotic or eukaryotic hosts. The term "transformation" of a cellular organism with DNA means the introduction of DNA into an organism in such a way that the DNA is replicable, either as an extrachromosomal element or by means of chromosomal integration. The term "transfection" of a cellular organism with DNA refers to the re-entry of DNA, for example, an expression vector, by means of the cell or organism if one or the other coding sequence is actually expressed. The terms "transfected host cell" and "transformed" refer to a cell in which DNA has been introduced. The cell is called a "host cell" and can be prokaryotic or eukaryotic. Typical prokaryotic host cells include various strains of E. coli. Eukaryotic host cells are of mammalian origin, such as, for example, derived from Chinese hamster ovary or cells of human origin. The introduced DNA sequence may come from the same species as the host cell of a species other than the host cell, or it may be a hybrid DNA sequence, that contains some strange DNA or some homologous DNA. The term "vector" means a DNA construct that contains a DNA sequence that is operably linked to a suitable control sequence capable of effecting expression of the DNA in a suitable host. These control sequences include a promoter to effect transcription, an optional operator sequence to control this transcription, a sequence encoding suitable mRNA-ribosome binding sites, and sequences that control the termination of transcription and translation. The vector can be a plasmid, a phage particle, or simply a potential genomic insert. Once transformed into a suitable host, the vector can replicate and function independently of the host genome, or it can sometimes be integrated into its genome. In the present specification, the terms "plasmid" and "vector" are sometimes used interchangeably, since the plasmid is the most commonly used form of vector. However, the invention is intended to include these other forms of vectors that have an equivalent function and which are, and may become, known in the art. The term "control sequences" refers to DNA sequences necessary for the expression of a coding sequence operably linked in a particular host organism. The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. It is known that eukaryotic cells use promoters, polyadenylation signals and enhancers. The DNA for a secretory leader or presequence can be operably linked to the DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it affects transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is located in order to facilitate translation. In general terms, the term "operatively linked" means that the ligated DNA sequences are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, the intensifiers do not have to be contiguous. The term "mammal" for the purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, non-human primates, and animals from zoos, competition or pets, such as dogs , horses, cats, cows, etc. The term "tagged epitope", when employed in the context of a polypeptide, refers to a polypeptide fused to an "epitope tag". The epitope tag polypeptide has enough residues to provide an epitope against which an antibody can be made, however it is fairly short so that it does not interfere with the activity of the polypeptide. The epitope tag of preference is also reasonably unique, such that the antibody practically does not cross-link with other epitopes. Suitable tag polypeptides usually have at least 6 amino acid residues and usually have between about 8-50 amino acid residues (preferably between about 9-30 residues). Examples include the FL HA tag polypeptide and its 12CA5 antibody (Field et al, 8: 2159-2165 (1998))); the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies against it (Evan et al., Mol Cell, Biol. 5 (12): 3610-3616 (1985)); and the Herpes Simplex Virus glycoprotein D (gD) label and its antibody (Paborsky et al., Protein Engineering 3 (6): 547-553 (1990)). In certain embodiments, the epitope tag may be an epitope of the FC region of an IgG molecule (eg, IgG1, IgG2, IgG3 or IgG4) that is responsible for increasing the average life of the serum in vivo of the IgG molecule. The word "tag", when used in this description, refers to a detectable compound or composition that can be directly or indirectly conjugated to a molecule or protein, for example, an antibody. The label itself is detectable (eg, radioisotope tags or fluorescent tags) or, in the case of an enzymatic tag, it can catalyze the chemical alteration of a detectable composition or substrate compound. As used herein, the term "solid phase" means a non-aqueous matrix to which the antibody of the present invention can adhere. The solid phase example encompassed in this disclosure includes those formed partially or completely of glass (e.g., controlled pore glass), polysaccharides (e.g., agarose), polyacrylamides, polystyrene, polyvinyl alcohol, and silicones. In certain embodiments, depending on the context, the solid phase may comprise the well of a test plate; in other contexts, it is a purification column (for example, an affinity chromatography column). As used in this description, the term "IgE mediated disorder" means a condition characterized by overproduction and / or hypersensitivity to immunoglobulin.

IgE Specifically, this term should be considered to include conditions associated with anaphylactic hypersensitivity and atopic allergies, including, but not limited to, asthma, rhinitis, and allergic conjunctivitis. (hay fever), eczema, urticaria, atopic dermatitis and food allergies. The serious physiological condition of anaphylactic shock caused for example, by bee stings, snake bites, food or medication, is also covered under the scope of this term.

Generation of antibodies The raw material antibody or "parent" can be prepared using techniques available in the art to generate this type of antibodies. These techniques are of general knowledge. Illustrative methods for generating the raw material antibody are described in greater detail in the following sections. These descriptions are possible alternatives for the preparation or selection of a parent antibody and in no way limit the methods by means of which this molecule can be generated. The binding affinity of the antibody is determined before the generation of a high affinity antibody of the present invention. Also, the antibody can be subjected to other assays of biological activity, for example, in order to evaluate efficacy as a therapeutic. This type of assays are in the knowledge of the technique and depend on the objective and the intended use for the antibody. To screen for antibodies that bind to a particular epitope (for example, those that block the binding of IgE with its high affinity receptor), a routine cross-blockade test, such as the one described in the work, can be carried out. : "Antibodies: A Laboratory Manual" (Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988)). Alternatively, epitope mapping can be performed to determine where the antibody binds to an epitope of interest. Optionally, the binding affinity of the antibody to a homolog of the target used to generate the antibody (where the homologue comes from a different species) can be assessed using techniques known in the art. In a modality, the other species are a non-human mammal to which the antibody will be administered in preclinical studies. Therefore, the species can be a non-human primate, such as, for example, Indian monkey, crab-eating macaques, baboon, chimpanzee and macaque. In other modalities, the species may be, for example, a rodent, cat or dog. The parent antibody is altered according to the present invention, in order to generate an antibody having a greater or more potent binding affinity towards the target compared to the parent antibody. The specificity of the antibody produces the unique interface that is formed between the antibody and its target; the surfaces complement each other to produce a unique accommodation (Jones, S. &Thornton, J. M. (1996) Proc. Nati, Acad. Sci. USA 93: 13-20). By further improving the contacts along this interface, the global affinity may increase as a result of the lower energy cost necessary to favor the association of the union partners. The binding surface of the antibody is composed, in general terms, by six complementarity determining regions (CDRs) that are loops that extend out from the nucleus. CDRs are composed of amino acids that have a sequence that is unique for binding to the specific target. To increase the affinity of an antibody to its antigen, the environment around these amino acids must be made more favorable by introducing or improving several non-covalent forces, which ultimately reduce the interaction energy, which produces a greater affinity. Van der Waals forces are non-covalent interactions that occur between two electrically neutral molecules (Voet, D. &Voet, J. G. (1990) Biochemistry John Wiley and Sons, NY, NY). The associations can be presented between two surfaces from the electrostatic interactions that originate from the permanent or induced dipoles. These dipoles may exist along the ends of nearby a-helices or polar amino acids. By increasing the number of Van der Waals forces along a binding interface, a more favorable association will occur. The introduction of hydrogen bonds will also increase the specificity of an interaction between an antibody and its antigen. The donors and acceptors involved in the bond are: nitrogen, oxygen and sulfur atoms. Whose amino acids are predominantly compounds (See Voet, et al., Supra). Hydrogen bonds tend to cross only short distances (usually 2.7 to 3.1 A), therefore the joint partners must be in close proximity for these interactions to occur. Accordingly, one way by which it can be improved is to place molecules of potential donors or acceptors in close contact to establish hydrogen bonds. Finally, improving hydrophobic interactions will also increase the favorable energetics between two binding partners. The non-polar debris that lies near the junction surface must be surrounded by other non-polar debris, and therefore, will exist in a favorable environment. By satisfying the cancellation of the non-polar secondary chains, the interaction energy is favorable for a more powerful binding interface. The interactions that stabilize the protein-protein interface reduce the energy cost of maintaining these contacts and consequently the overall affinity will increase. By improving the environment around the individual amino acids that are close to the binding interface, a more favorable environment is produced which results in a higher binding affinity. Therefore, by introducing favorable contacts and by improving the interface through additional supplementation, the overall binding interaction between the antibody and the antigen will be considerably improved. The resulting high affinity antibody preferably has a target binding affinity of at least about 10 times greater, or at least about 20 times greater, or at least about 500 times greater or may be 1000 to 5000 times greater than the binding affinity of the parent antibody, towards the target. The degree of improvement in the binding affinity required or desired will depend on the initial binding affinity of the parent antibody. In general, the method for making high affinity antibodies from a parent antibody involves the following steps: 1. Obtain or select a parent antibody that binds to the target of interest, which comprises variable domains of light chain or heavy chain. This can be carried out by traditional hybridoma techniques, phage display techniques or any other method for the generation of a specific antibody to a target. 2. Select a sequence frame structure similar to the parent framework, preferably a human template sequence. This mold can be chosen based on, for example, its overall comparative length, the size of the CDRs, the amino acid residues located at the junction between the framework and the CDRs, the global homology, etc. The chosen mold can be a mixture of more than one sequence or it can be a consensus mold. 3. Generate a clone library by making random amino acid substitutions in each and every one of the possible positions of the CDRs. Someone could also substitute the amino acids in the template of human framework that are, for example, adjacent to the CDRs or that can affect the union or representation, with all the possible amino acids, generating a library of framework substitutions. These framework substitutions can be evaluated for their potential effect on binding to the target and on the presentation of antibodies. The substitution of amino acids in the framework can be carried out either simultaneously or sequentially with the substitution of amino acids in the CDRs. One method for the generation of the variant library is by the synthesis of oligonucleotides. 4. Construct an expression vector comprising the heavy chain and / or light chain variants generated in step (3) which comprise the formulas: FRH1-CDRH1-FRH2-CDRH2-FRH3-CDRH3-FRH4 (I) and FRL1 -CDRL1-FRL2-CDRL2-FRL3-CDRL3-FRL4 (II), wherein FRL1, FRL2, FRL3, FRL4, FRH1, FRH2, FRH3 and FRH4 represent the variants of the light chain and heavy chain sequences of the selected framework mold in step 3 and the CDRs represent the CDRs variant of the CDRs of the parent antibody. An example of a vector containing these light chain and heavy chain sequences is described in Figure 1. 5. Screening the clone library against the specific target, and those clones that bind to the target are screened for an improved binding affinity. . Those clones that bind with higher affinity than the progenitor molecule can be selected. The optimal high affinity candidate will have the highest possible affinity compared to the parent antibody, preferably greater than 20 times, 100 times, 1000 times or 5,000 times. If the chosen variant contains certain amino acids that are undesirable, such as an introduced glycosylation site or a potentially immunogenic site, these amino acids can be replaced with more beneficial amino acid residues and the binding affinity can be re-evaluated. Someone can also use this method to generate high affinity antibodies from a completely human parent antibody by randomly replacing only the CDR regions, leaving the human framework intact. Due to improved high-throughput screening techniques and vectors, such as those described in Figure 1, a technician can quickly and efficiently screen a comprehensive substitution library at all sites in a given CDR and / or framework region. By randomly substituting all amino acids at all positions simultaneously, one is able to screen for possible combinations that greatly increase the affinity that would not have been anticipated or identified by individual substitution due, for example, to synergy.

PREPARATION OF THE PROGENITOR ANTIBODY Preparation of the target Soluble targets or fragments thereof can be used as immunogens to generate antibodies. The antibody is directed against the target of interest. Preferably, the target is a biologically important polypeptide and administration of the antibody to a mammal suffering from some disease or disorder can produce a therapeutic benefit in that mammal. However, antibodies can be directed against non-polypeptide targets. When the target is a polypeptide, it can be a transmembrane molecule (eg, a receptor) or a ligand, such as a growth factor. An objective of the present invention is IgE. Complete cells can be used as the immunogen for the preparation of antibodies. The target can be produced recombinantly or can be made using synthetic methods. The target can also be isolated from a natural source. The antigens used in the production of antibodies of the present invention can include polypeptides and fragments of polypeptides of the invention, including epitope A and / or epitope B. A polypeptide used to immunize an animal can be obtained by standard recombinant, synthetic chemical methods or purification. As is well known in the art, in order to increase immunogenicity, an antigen can be conjugated with a carrier protein. Carriers that are commonly used include, but are not limited to, keyhole limpet hemocyanin (KLH), thyroglobulin, bovine serum albumin (BSA, Bovine Serum Albumin), and tetanus toxoid. The coupled peptide is then used to immunize an animal (e.g., a mouse, a rat or a rabbit). In addition to this type of carriers, well-known adjuvants can be administered with the antigen in order to facilitate the induction of a potent immune response.

Polyclonal antibodies Polyclonal antibodies are usually generated in non-human mammals by multiple utaneous (se) or intraperitoneal (ip) injections of the relevant target in combination with an adjuvant. Many agents capable of producing an immune response are known in the art. The animals were immunized against the target, immunogenic conjugates or derivatives by combining the protein or conjugate (for rabbits or mice, respectively) with complete Freund's adjuvant and by injecting the solution intradermally. One month later, animals were supercharged with 1/5 to 1/10 of the original amount of polypeptide or conjugate in Freund's complete adjuvant by utaneous injection at multiple sites. From 7 to 14 days later, the animals were fed and the serum was titrated for antibody titration. The animals were taken to the assessment plates. The mammalian antibody selected by the usual will have a sufficiently strong binding affinity towards the target. For example, the antibody can bind the human anti-IgE target with a binding affinity value (Kd) of about 1 x 10"8 M. The affinities of the antibody can be determined by saturation binding Enzyme-Linked Immunoenzymatic Assay (ELISA, Enzyme linked immunos or rbent assay), and competition assays (eg, radioimmunoassays). For the screening of antibodies that bind to the target of interest, a routine crosslinking assay may be carried out, for example , which is described in the work: Antibodies, A Laboratory Manual, Cold Spring Harbor Labora tory, Ed and David Lane (1988) .Alternatively, in order to determine the union epitope mapping can be carried out, for example, as described in Champe, et al., J. Biol. Chem. 270: 1388-1394 (1995).

Monoclonal antibodies Monoclonal antibodies are antibodies that recognize a single antigenic site. Its uniform specificity produces much more useful monoclonal antibodies than polyclonal antibodies, which usually contain antibodies that recognize a variety of different antigenic sites. Monoclonal antibodies can be made using the hybridoma method first described by Kohier et al., Nature, 256: 495 (1975), or they can be made by recombinant DNA methods. In the hybridoma method, a mouse or other suitable host animal, such as a rodent, is immunized as described above to obtain lymphocytes that produce or are capable of producing antibodies that will bind specifically to the protein used. for immunization. Alternatively, lymphocytes can be immunized in vitro. The lymphocytes are then fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, for the purpose of forming a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). Hybridoma cells prepared in this manner are seeded and allowed to grow in a suitable culture medium which preferably contains one or more substances that inhibit the growth or survival of the unfused and progenitor myeloma cells. For example, if the progenitor myeloma cells lack the enzyme hypoxanthine-guanine phosphoribosyltransferase (Hypoxanthine Guanine Phophoribosyl Transf erase, HGPRT or HPRT), the culture medium for the hybridomas will usually include hypoxanthine, aminopterin and thymidine. (medium HAT), substances that prevent the growth of cells deficient in HGPRT. Preferred myeloma cells are those that fuse efficiently, support a stable high-level production of antibodies by means of the selected cells producing the antibody, and are sensitive to a medium, such as, for example, the HAT medium. The human myeloma and mouse-human heteromyeloma cell lines have been described for the production of human monoclonal antibodies (Kozbar, J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc. New York, 1987)). After hybridoma cells are identified that produce antibodies of specificity, affinity and / or desired activity, clones can be subcloned by limiting dilution and growth procedures by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pgs. 59-103 (Academic Press, 1986)). Suitable culture media for this purpose include [sic]. The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium by standard immunoglobulin purification procedures, such as, for example, protein A-Sepharose chromatography, hydroxylapatite chromatography, gel electrophoresis, dialysis or affinity chromatography. The DNA encoding the monoclonal antibodies is easily isolated and sequenced using conventional methods (for example, using oligonucleotide probes that are capable of binding specifically to genes encoding the light chain and heavy chain of the monoclonal antibodies). Hybridoma cells serve as a source of this type of DNA. Once isolated, the DNA can be placed in expression vectors, which are then transferred into host cells such as E cells. coli, NSO cells, Chinese hamster ovarian cells (CHO), myeloma cells, in order to obtain the synthesis of monoclonal antibodies in recombinant host cells. DNA can also be modified, for example, by substituting the coding sequence for the human light chain and heavy chain constant domains of murine homologous sequences (U.S. Patent No. 4,816,567; Morrison et al., Proc. Nati Acad. Sci. USA 81: 6851 (1984)), or by covalently binding to the immunoglobulin polypeptide.

Humanized Antibodies Humanization is a technique for obtaining a chimeric antibody, wherein considerably less than an intact human variable domain has been replaced by the corresponding sequence from a non-human species. A humanized antibody has one or more amino acid residues introduced therein from a source; which is not human. These non-human amino acid residues are often referred to as "import" residues, which usually come from the "import" variable domain. Humanization can be carried out following basically the method of Winter et al. (Jones et al, Nature 321: 522-525 (1986); Riechman et al., Nature 332: 323-327 (1988); Verhoeyens et al., Verhoeyens et al., Science 239: 1534-1536 (1988)), by substituting the CDRs or non-human CDR sequences for the corresponding sequences in a human antibody (See, for example, U.S. Patent No. 4,816,567). In practicing the present invention, the humanized antibody may have some CDR residues and some FR residues substituted with residues from analogous sites in murine antibodies. The choice of human variable domains, both light chain and heavy chain, for use in the preparation of humanized antibodies is very important to reduce antigenicity. According to the method called "best accommodation", the sequence of the variable domain of a non-human antibody is compared to the library of the known human variable domain sequences. The human sequence that is closest to that of the non-human parent antibody is then accepted as the human framework for the humanized antibody Sims et al., J. Immunol. 151: 2296 (1993); Chothia et al., J. Mol. Biol. 196: 901 (1987)). Another method employs a particular lattice derived from the consensus sequence of all human antibodies of a particular subgroup of light chains or heavy chains. The same framework can be used for various humanized antibodies (Cárter et al., Proc Nati Acad Sci USA, 89: 4285 (1992), Presta et al., J. Immunol. 151: 2623 (1993)).

Antibody fragments Various techniques have been developed for the production of antibody fragments. Typically, these fragments were derived via proteolytic digestion of the intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24: 107-117 (1992) and Brennan et al., Science 229: 81 (1985)). However, these fragments can now be produced directly by recombinant host cells. For example, antibody fragments can be isolated from antibody phage library. Alternatively, F (ab ') 2-SH fragments can be recovered directly from E. coli and can be chemically coupled to form F (ab') 2 fragments (Carter et al., Bio / Technology 10: 163 -167 (1992)). According to another methodology, F (ab ') 2 fragments can be isolated directly from culture of recombinant host cells. Other techniques for the production of antibody fragments will be apparent to the skilled artisan. In other embodiments, the antibody of choice is a single chain Fv fragment (scFv). (PCT patent application WO 93/16185).

PREPARATION OF HIGH AFFINITY ANTIBODIES Once the parent antibody has been identified and isolated, one or more amino acid residues can be altered in one or more variable regions of the parent antibody. Alternatively or additionally, one or more substitutions of framework residues can be introduced into the parent antibody, where they produce an improvement in the binding affinity of the antibody, for example, for human IgE. Examples of framework region residues to be modified include those that do not bind covalently or directly to the target (Amit et al., Science 233: 747-753 (1986)); interact with and / or affect the conformation of the CDR (Chothia et al., J. Mol. Biol. 196: 901-917 (1987)); and / or participate in the VL-VH interface (EP 239 400 Bl). In certain embodiments, modification of one or more of these framework region residues results in an improvement of the antibody binding affinity for the target of interest. Modifications to the biological properties of antibodies can be made by selecting substitutions that differ considerably in their effect on maintenance, for example, (a) the structure of the polypeptide backbone in the area of substitution, eg, as a sheet or helical conformation; (b) the charge or hydrophobicity of the molecule at the target site, or (c) the volume of the secondary chain. Non-conservative substitutions will involve the exchange of a member of one of these classes for one of another class.

The nucleic acid molecules encoding the amino acid sequence variants are prepared by a range of methods known in the art. These methods include, among others, oligonucleotide-mediated (or site-directed) mutagenesis, PCR (Polymerase chain reaction) mutagenesis, and cassette mutagenesis of a previously prepared variant or a non-variant version of the antibody dependent on the species. The preferred method for the generation of variants is an oligonucleotide-mediated synthesis. In certain embodiments, the antibody variant will only have a residue of a single hypervariable region, for example, from about two to about fifteen substitutions of hypervariable regions. One method for generating the library of variants is by oligonucleotide-mediated synthesis according to the scheme described in Figure 2. Each of three oligonucleotides of about 100 nucleotides can be synthesized by spreading the complete region of the complete light chain or heavy chain . Each oligonucleotide may comprise: (1) a stretch of 60 amino acids generated by the triplet (NNK) 20, where N is any nucleotide and K is G or T, and (2) an overlap of about 15-30 nucleotides either with the next oligo and with the vector sequence at each end. With the pairing of these three oligonucleotides in a PCR reaction, the polymerase will be filled in the opposite strand generating a complete sequence of variable region of heavy chain or light chain of double strand. The number of triplets can be adjusted to any length of repeats and their position within the oligonucleotide can be chosen to only substitute amino acids in a given CDR or framework region. By employing (NNK), all twenty amino acids in each position are possible in the coding variants. The overlapping sequence of 5-10 amino acids (15-30 nucleotides) will not be substituted, but it can be chosen to be within the stacking regions of the framework, or it can be replaced by a separate or consecutive synthesis cycle. Methods for synthesizing oligonucleotides are generally known in the art and are also commercially available. Methods for the generation of antibody variants from these oligonucleotides are also generally known in the art, for example, the PCR technique. The library of heavy chain and light chain variants, which differ in random positions in their sequences, can be constructed in any expression vector, such as, for example, the bacteriophage, specifically the vector of Figure 1, each of which contains DNA that encodes a heavy chain or light chain variant in particular. After the production of the antibody variants, the biological activity of the variant with respect to the parent antibody is determined. As noted above, this involves determining the binding affinity of the variant for the target. There are numerous high throughput methods for the rapid screening of antibody variants for their ability to bind to the target of interest. One or more antibody variants selected from this initial screening can then be screened for their improved binding affinity to the parent antibody. A common method for the determination of binding affinity is to assess the association and dissociation index constants using a BIAcore ™ surface plasmon resonance system (BIAcore, Inc.). A biosensor chip is activated for the covalent coupling of the lens, according to the manufacturer's instructions (BIAcore). The target is then diluted and injected onto the chip to obtain a signal in response units (RU) of the immobilized material. Since the signal in RU is proportional to the mass of the immobilized material, it represents a range of target densities immobilized in the matrix. Dissociation data are fixed to a mold of a site to obtain Kdesacopie + / ~ s.d. (standard deviation of measurements). The constant (Ks) of first order proportion for association curve is calculated and plotted as a function of the protein concentration to obtain the Kacop? E +/- (see standard error of fit). The equilibrium dissociation constants for the bond, KD, are calculated from SPR measurements such as Kdesacople / KaCopie. Since the equilibrium dissociation constant, KD, is inversely proportional to Kesacopie? an affinity improvement estimate can be made assuring the association (Kac? foot) is a constant for all variants. The resulting candidate or candidates with high affinity may optionally be subjected to one or more additional assays of biological activity in order to confirm that the variant or variants of antibody with enhanced high affinity still maintain the desired therapeutic attributes. For example, in the case of an anti-IgE antibody, someone can screen for those that block the binding of IgE with its receptor and inhibit the release of histamine. The optimal antibody variant maintains the ability to bind to the target with a considerably higher binding affinity compared to the parent antibody.

Frequently, the antibody (s) thus selected may be subjected to further modifications depending on the intended use of the antibody. This type of modifications may involve the additional alteration of the amino acid sequence, its fusion with one or more heterologous polypeptides and / or covalent modifications, such as, for example, those elaborated below. For example, any cysteine residue not involved in the preservation of the correct conformation of the antibody variant, usually with serine, can be substituted in order to improve the oxidative stability of the molecule and in order to avoid cross-linking anomalous On the other hand, one or more cysteine bonds can be added to the antibody to improve its stability (particularly when the antibody is an antibody fragment, such as, for example, the Fv fragment).

VECTORS The invention also provides isolated nucleic acid encoding an antibody variant as described herein, vectors and host cells comprising the nucleic acid, and recombinant techniques for the production of the antibody variant. For the recombinant production of the antibody variant, the encoding nucleic acid is isolated and inserted into a replicable vector for further cloning (amplification of the DNA) or for its expression. The DNA encoding the antibody variant is isolated and easily sequenced using conventional methods (eg, the use of oligonucleotide probes that are capable of specifically binding to genes encoding the light chains and heavy chains of the antibody variant). There are many vectors available. The vector components include, in general terms, but not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter and a terminator sequence. transcription. The phage vector of expression described in Figure 1 consists of a commonly used M13 vector and the viral secretion signal of gene III of M13 for the Fab variant of screening and rapid secretion for the correct binding specificity and for the minimum affinity criteria. This vector does not use the complete sequence of gene III, therefore it is not shown on the surface of the bacterial cell, but Fab is secreted in the periplasmic space. Alternatively, Fabs could be expressed in the cytoplasm and could be isolated. Each of the light and heavy chains has its own viral secretory signal, but they are expressed independently from a single potent inducible promoter. The vector in Figure 1 also provides a His tag and a Myc tag to facilitate purification, as well as detection. A skilled artisan would recognize that the Fabs could be expressed independently from separate promoters or that it is not necessary for the secretion signal to be the chosen viral sequence, but could be a prokaryotic or eukaryotic signal sequence suitable for the secretion of the fragments of antibody from the chosen host cell. It should also be noted that light and heavy chains can reside in different vectors.

A. Component of the signal sequence The antibody variant of this invention can be produced recombinantly. The variant can also be expressed as a fusion polypeptide fused to a heterologous polypeptide, which is preferably a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature polypeptide or protein. The heterologous signal sequence selected is preferably one that is recognized and processed (i.e., cleaved by means of a signal peptidase) by the host cell. For prokaryotic host cells that do not recognize and process the signal sequence of the native antibody, the signal sequence can be replaced by a prokaryotic signal sequence selected, for example, from the group of the leaders of alkaline phosphatase, penicillinase, Ipp. or thermostable enterotoxin II. 0 in the case of the vector of Figure 1, the signal sequence chosen was a viral signal sequence from gene III. For yeast secretion, the native signal sequence can be substituted by, for example, the leader of yeast invertase, the leader of a-factor (including the leaders of the a-factors of Saccharomyces and Kluyveromyces) or the leader of acid phosphatase, the glucoamylase leader of C. albicans, or by a signal described for example, in WO 90/13646. In the expression of mammalian cells, mammalian signal sequences as well as secretory leaders are available, for example, the gD signal of herpes simplex. The DNA for this precursor region is ligated in the reading frame to the DNA encoding the antibody variant.

B. Origin of replication component Usually, the vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells. In general, this sequence is one that enables the vector to replicate independently of the chromosomal DNA of the host, and includes origins of replication or autonomously replicating sequences. These types of sequences are well known for a range of bacteria, yeasts and viruses. The origin of replication from plasmid pBR322 is suitable for most gram-negative bacteria, the 2μ plasmid origin is suitable for yeast, and several viral origins (SV40, polyoma, adenovirus, VSV or BPV) are useful for vectors in mammalian cells. In general, the origin of the replication component is not necessary for the mammalian expression vectors (the SV40 origin can be used, usually, only because it contains the initial promoter).

C. Selection of the genetic component Vectors may contain a selection gene, also called a selectable marker. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, eg, ampicillin, neomycin, methotrexate or tetracycline, (b) complement autotrophic deficiencies, or (c) supply crucial nutrients not found in media complexes, for example, the gene encoding D-alanine racemase for Bacilli. An illustrative selection scheme employs a drug to stop the growth of a host cell. Those cells that are successfully transformed with a heterologous gene produce a protein that confers drug resistance and thereby survive the selection regimen. Examples of this type of dominant selection employ the drugs neomycin, mycophenolic acid, and hygromycin. Another example of selectable markers suitable for mammalian cells are those that enable the identification of cells competent to absorb the antibody nucleic acid, such as, for example, DHFR (dihydrofolate reductase), thymidine kinase, metallothionein-I and II, preferably metallothionein genes. of primates, adenosine deaminase, ornithine decarboxylase, etc. For example, cells transformed with the DHFR selection gene are first identified by culturing all transformants in a culture medium containing methotrexate (Mtx), a competitive antagonist of DHFR. A suitable host cell when the wild-type DHFR employed is the Chinese hamster ovary (CHO) cell line deficient in DHFR activity. Alternatively, host cells (in particular wild-type hosts containing endogenous DHFR) transformed (ATCC 20,622 or 38,626) or cotransformed with DNA sequences encoding the antibody, wild-type DHFR protein, can be selected another selectable marker , such as, for example, aminoglycoside 3'-phosphotransferase (APH, aminoglycoside 3'-phosphotransf erase) by cell growth in a medium containing a selection agent for the selectable marker, such as, for example, an aminoglycoside antibiotic, e.g., neomycin or G418 (U.S. Patent No. 4,965,199). A suitable selection gene for use in yeast is the trp 1 gene in yeast plasmid Yrp7 (Stinchcomb et al., Nature 282: 39 (1979)). The trp 1 gene provides a selection marker for a variant strain of yeast lacking the ability to grow in tryptophan, 'e.g., ATCC No. 44016 or PEP4-1. Jones, Genetics 85: 12 (1977). The presence of trp 1 lesion in the genome of the yeast host cell provides an efficient improvement to detect transformation by growth in the absence of tryptophan. Similarly, Leu-2 deficient yeast strains (ATCC 20,622 or 38,626) are supplemented with known plasmids carrying the Leu2 gene.

D. Promoter Component The expression and cloning vectors usually contain a promoter which is recognized by the host organism and which is operably linked to the antibody nucleic acid. Promoters suitable for use with prokaryotic hosts include the phoA promoter, β-lactamase and lactose promoter systems, alkaline phosphatase, a tryptophan (trp) promoter system, and hybrid promoters, such as, for example, the tac promoter. However, other known promoters are suitable. Promoters for use in bacterial systems may also contain a Shine-Dalgarno (S.D.) sequence operably linked to the DNA encoding the antibody. Promoter sequences for eukaryotes are known. Virtually all eukaryotic genes have an AT-rich region located approximately 25 to 30 bases upstream of the site where transcription starts. Another sequence found 70 to 80 bases upstream of the start of transcription of many genes is a CNCAAT region, where N can be any nucleotide. At the 3 'end of most eukaryotic genes is an ATA sequence which may be the signal for the addition of the poly A tail to the 3' end of the coding sequence. All sequences are suitably inserted into eukaryotic expression vectors. Examples of suitable promoter sequences for use with yeast hosts include promoters for 3-phosphoglycerate kinase or other glycolytic enzymes, such as, for example, enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate. isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase and glucokinase. Other promoters of yeasts, which are inducible promoters that have the additional advantage of controlled transcription by growth conditions, are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degrading enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde- 3-phosphate dehydrogenase, and enzymes responsible for the use of maltose or galactose. Suitable vectors and promoters for use in yeast expression have been further described in EP 73,657. Yeast enhancers are used to advantage with yeast promoters. Antibody transcription is controlled from vectors in mammalian host cells, for example, by promoters obtained from the genomes of viruses, such as, for example, polyoma virus, fowl pox virus (fowlpox), adenovirus (e.g. , Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis B virus and, more preferably, Simian virus 40 (SV40), also obtained from heterologous mammalian promoters, for example, the actin promoter or an immunoglobulin promoter, also obtained from heat shock promoters, as long as these promoters are compatible with host cell systems. The early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment that also contains the SV40 viral origin of replication. The immediate early promoter of human cytomegalovirus is conveniently obtained as a HindIII E restriction fragment. No. 4, 419,446 describes a system for the expression of DNA in mammalian hosts using the bovine papilloma virus as a vector. In the U.S. patent No. 4,601,978 describes a modification of this system. Alternatively, human β-interferon cDNA has been expressed in mouse cells under the control of a thymidine kinase promoter from the herpes simplex virus. On the other hand, long terminal repeats of the Rous sarcoma virus can be used as the promoter.

E. Component of the enhancer element The transcription of a DNA encoding the antibody of this invention by eukaryotes is often increased by the insertion of an enhancer sequence into the vector. Many intensifying sequences are known from mammalian genes (globin, elastase, albumin, α-fetoprotein and insulin).

However, in general, someone will use an intensifier derived from a eukaryotic cell virus.

Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the enhancer of the cytomegalovirus early promoter, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. See also Yaniv, Nature 297: 17-18 (1982) in enhancing elements of eukaryotic promoter activation. The enhancer can be cut and spliced in the vector at a position 5 'or 3' to the coding sequence of the antibody, but preferably located at a 5 'site from the promoter.

F. Transcription Termination Component Expression vectors employed in eukaryotic host cells (yeast, fungal, insect, plant, animal, human or nucleated cells derived from other multicellular organisms) may also contain sequences necessary for the termination of transcription and for the stabilization of the mRNA. Such sequences are usually available from the 5 'untranslated regions, and sometimes from the 3' untranslated regions of eukaryotic or viral DNA or cDNA. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding the antibody. A useful transcription termination component is the polyadenylation region of bovine growth hormone. See, for example, WO 94/11026.

SELECTION AND TRANSFORMATION OF GUEST CELLS Suitable host cells for the cloning or expression of DNA in the vectors of the present invention are prokaryotic, yeast or higher eukaryotic cells. Prokaryotes suitable for this purpose include both gram-negative organisms as well as gram-positive organisms, eg, enterobacteria, such as, for example, E. coli, Enterobacter, Klebsiella, Proteus, Salmonella, Serra tia and Shigella, as well as Bacilli. , Pseudomonas and Streptomyces. A preferred E. coli cloning the host is E. coli 294 (ATCC 31,446), although other strains are suitable, such as, for example, E. coli B, E. coli Xlll 6 (ATCC 31,537) and E. coli W3110 ( ATCC 27,325). These examples are illustrative but not limiting. In addition to prokaryotes, eukaryotic microbes, such as, for example, yeast or filamentous fungi are suitable for the cloning or expression of hosts of vectors encoding the antibody. Saccharomyces cerevisiae is the most commonly used among lower eukaryotic host microorganisms. However, a range of other genera, species and strains are in common use and are useful for the present invention, such as, for example, Shizo saccharomyces pombe; Kluyveromyces; Candida; Trichoderma; Neurospora crassa; and filamentous fungus, such as, for example, hosts of Neurospora, Penicillium, Tolypociadium and Aspergillus, such as, for example, A. nidulans and A. niger. Host cells suitable for the expression of glycosylated antibodies are derived from multicellular organisms. In general, any eukaryotic cell culture is feasible, whether it is a vertebrate or invertebrate culture. Examples of invertebrate cells include plant and insect cells, Luckow et al., Bio / Technology 6, 47-55 (1988); Miller et al., Genetic Engineering, Setlow et al. eds. Vol. 8, pgs. 277-279 (Plenam, publication 1986); Mseda et al., Nature 315, 592-594 (1985). Numerous variants and baculoviral strains and corresponding permissive insect host cells have been identified from hosts, such as Spodoptera frugiperda (caterpillar), Aedes (mosquito), Drosophila (fruit fly) and Bombyx mori. A variety of viral strains for transfection are available to the public, for example, the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and these viruses can be employed as the virus of the present invention. , in accordance therewith, in particular for the transfection of Spodoptera frugiperda cells. Furthermore, plant cell cultures of cotton, corn, potato, soybean, petunia, tomato and tobacco are used as hosts. Vertebrate cells and their propagation in culture (tissue culture) have become routine. See the work: "Tissue Cul ture", Academic Press, Kruse and Patterson, eds. (1973). Examples of useful mammalian host cells are monkey kidney cells; human embryonic kidney cell line; baby hamster kidney cells; Chinese hamster ovary cells / -DHFR (CHO, Uriaub et al., Proc. Nati, Acad. Sci. USA 77: 4216 (1980)); Mouse Sertoli cells; human cervical carcinoma cells (HELA, human cervical carcinoma cell); canine kidney cells; human lung cell; human liver cells; mouse mammary tumor; and NSO cell: Host cells are transformed with the above described vectors for the production of antibodies and cultured in modified conventional nutrient media as appropriate for the induction of promoters, selection of transformants or for the amplification of the genes encoding the sequences desired. The host cells employed to produce the antibody variant of this invention can be cultured in a variety of media. For the cultivation of the host cells, the media available on the market are suitable, such as, for example, Ham's FIO (Sigma), Minimum Medium Essential (MEM, Minimal Essential Medium, Sigma), RPMI-1640 (Sigma) and half Eagle modified by Dulbecco (DMEM, Dulbecco 's Modified Eagle' s Medium, Sigma). In addition, any of the means described in Ham et al., Meth. Enzymol. 58: 44 (1979), Barnes et al., Anal. Biochem. 102: 255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,560,655; 5,122,469; 5,712,163; or 6,048,728 can be employed as the culture media for the host cells. Any of the means described can be supplemented, as necessary, with hormones and / or growth factors (such as insulin, transferrin or epidermal growth factor), salts (such as, for example, X-chlorides, where X is sodium , calcium, magnesium and phosphates) buffers (such as HEPES), nucleotides (such as, for example, adenosine and thymidine), antibiotics (such as the drug GENTAMYCIN. TM.), trace elements (defined as inorganic compounds usually present in final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplement in suitable concentrations that would be known to those skilled in the art may also be included. The culture conditions, for example, temperature, pH and the like, are those previously employed with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.

ANTIBODY PURIFICATION When recombinant techniques are used, the antibody variant can be produced intracellularly, in the periplasmic space, or it can be directly secreted in the medium. If the antibody variant is produced intracellularly, as a first step, debris can be removed from particles, either host cells or lysed fragments, for example, by centrifugation or ultrafiltration. Carter et al., Bio / Technology 10: 163-167 (1992) describe a method for isolating antibodies that are secreted into the periplasmic space of E. coli Briefly, the cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA and phenylmethylsulfonyl fluoride (PMSF) for about 30 minutes. The cellular debris can be removed by centrifugation. When the antibody variant is secreted into the medium, the supernatants of these expression systems are usually first concentrated using a commercially available protein concentration filter, an Amicon or Millipore Pellicon ultrafiltration unit. A protease inhibitor, such as PMSF, may be included in any of the above steps to inhibit proteolysis, and antibiotics may be included to prevent the growth of unexpected contaminants. The antibody composition prepared from the cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis and affinity chromatography; Affinity chromatography is the preferred purification technique. The suitability of protein A as an affinity ligand depends on the species and isotype of any Fc domain of the immunoglobulin that is present in the antibody variant. Protein A can be used to purify antibodies that are based on heavy chains IgG1, IgG2 or human IgG4 (Lindmark et al., J. Immunol Meth. 62: 1-13 (1983)). The G protein is recommended for all mouse isotypes and for human IgGE (Guss et al., EMBO J. 5: 1567: 1575 (1986)). The matrix to which the affinity ligand is linked in most cases in the agarose, but other matrices are available. Mechanically stable matrices, such as controlled pore glass or poly (styrenediyl) benzene, allow for faster flow rates and shorter processing times compared to those that can be achieved with agarose. When the antibody variant comprises a CH3 domain, the Bakerbond ABX ™ resin (J.T. Baker, Phillipsburg, N.J.) is useful for purification. Depending on the antibody variant to be recovered, other protein purification techniques are also available, such as fractionation in an ion exchange column, ethanol precipitation, reverse phase high performance liquid chromatography (HPLC), chromatography in silica, SEPHAROSE ™ heparin chromatography, chromatography on an anionic or cationic exchange resin (such as polyaspartic acid column), chromatofocusing, SDS-PAGE and precipitation with ammonium sulfate. Following either the preliminary purification step or steps, the mixture comprising the antibody variant of interest and the contaminants can be subjected to hydrophobic interaction chromatography at a low pH using an elution buffer at a pH between about 2.5-4.5, preferably, made at low salt concentrations (eg, about 0-0.25M salt salt).

PHARMACEUTICAL FORMULATIONS The pharmaceutical formulations of the polypeptide or antibody can be prepared to be stored as lyophilized formulations or aqueous solutions by mixing the polypeptide having the desired degree of purity with "pharmaceutically acceptable" carriers, excipients, or stabilizers which are usually used in the technique (all are called "excipients"). For example, buffering agents, stabilizing agents, preservatives, isotonifiers, non-ionic detergents, antioxidants and other miscellaneous additives. (See Remington's Pharmaceutical Sciences, Sixteenth Edition, A. Osol, Ed. (1980) This type of additives must be toxic to the containers at the dosages and concentrations used The buffering agents help keep the pH in the range Approaching physiological conditions are present in a concentration range of about 2 mM to 50 mM Suitable buffering agents for use with the present invention include both organic and inorganic acids and their salts, such as, for example, buffering agents. citrate (for example, mixture of monosodium citrate-disodium citrate, mixture of citric acid-trisodium citrate, mixture of citric acid-monosodium citrate, etc.), succinate buffering agents (for example, mixture of succinic acid-monosodium succinate, mixture of succinic acid-sodium hydroxide, mixture of succinic acid-disodium succinate, etc.), tartrate buffers (for example, mixture of tartaric acid-sodium tartrate, mixture of tartaric acid-potassium tartrate, mixture of tartaric acid-sodium hydroxide, etc.), fumarate-deadening agents (for example, mixture of fumaric acid-monosodium furamate, etc.). ), fumarate-deadening agents (for example, mixture of fumaric acid-monosodium furamate, mixture of fumaric acid-disodium furamate, mixture of monosodium furamate-disodium furamate, etc.), gluconate-absorbing agents (eg, gluconic acid mixture) sodium gluconate, mixture of gluconic acid-sodium hydroxide, mixture of gluconic acid-potassium gluconate, etc.), oxalate buffering agents (eg, oxalic acid-sodium oxalate mixture, oxalic acid-sodium hydroxide mixture, oxalic acid potassium oxalate, etc.), lactate buffering agents (for example, lactic acid-sodium lactate, lactic acid-sodium hydroxide mixture, lactic acid mixture, potassium-lactate, etc.) and acetate buffering agents (eg, acetic acid-sodium acetate, acetic acid-sodium hydroxide, etc.). Additionally, mention may be made of phosphate buffering agents, histidine buffering agents and trimethylamine salts, such as, for example, Tris. Conservatives can be added to retard microbial growth, and can be added in amounts ranging from 0.2% -l% (w / v). Suitable preservatives for use with the present invention include phenol, benzyl alcohol, meta-cresol, methyl paraben, propyl paraben, octadecyldimethylbenzyl ammonium chloride, benzalkonium halides, benzalkonium halides (eg, chloride, bromide, iodide), hexamethonium, alkyl parabens, such as, for example, methyl or propyl paraben, catechol, resorcinol, cyclohexanol and 3-pentanol. Isotonicizers known as "stabilizers" can be added to ensure the isotonicity of liquid compositions of the present invention and include polyhydric sugar alcohols, preferably trihydric alcohols or higher sugars, such as, for example, glycerin, erythritol, arabitol, xylitol, sorbitol and mannitol The term "stabilizers" refers to a broad category of excipients that can range from a bulking agent to an additive that solubilizes the therapeutic agent or helps to avoid denaturation or adhesion in the wall of the container. Typical stabilizers may be sugar polyhydric alcohols (listed above); amino acids, such as, for example, arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, threonine, etc., organic sugars or sugar alcohols, such as, for example, lactose , trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, myoinisitol, galactitol, glycerol and the like, including cyclitol, such as, for example, inositol; polyethylene glycol; amino acid polymers; sulfur-containing reducing agents, such as, for example, urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, α-monothioglycerol and sodium thiosulfate; low molecular weight polypeptides (ie. <10 residues); proteins, such as, for example, human serum albumin, bovine serum albumin, gelatin or immunoglobulins; hydrophilic polymers, such as, for example, monosaccharides of polyvinyl pyrrolidone, such as, for example, xylose, mannose, fructose, glucose; disaccharides, such as, for example, lactose, maltose, sucrose; trisaccharides, such as, for example, raffinose; polysaccharides, such as, for example, dextran. Stabilizers may be present in the range of 0.1 to 10, 000 pesos per part of the active protein weight. Non-ionic surfactants or detergents (also known as "wetting agents") can be added to help stabilize the therapeutic agent, as well as to protect the therapeutic protein against agitation-induced aggregation, which also allows the formulation to be exposed to a Outstanding cutting surface without causing denaturation of the protein. Suitable nonionic surfactants include polysorbates (20, 80, etc.), polyoxamers (184, 188, etc.), Pluronic® polyols, polyoxyethylene sorbitan monoethers (TWEEN®-20, TWEEN®-80, etc.). Nonionic surfactants may be present in a range from about 0.05 mg / ml to 1.0 mg / ml, preferably from about 0.07 mg / ml to 0.2 mg / ml. Additional miscellaneous excipients include bulking agents, (e.g., starch), chelating agents (e.g., EDTA), antioxidants (e.g., ascorbic acid, methionine, vitamin E) and cosolvents. The formulation of the present invention may also contain more than one active compound, as necessary, for the particular indication to be treated, preferably those with complementary activities that do not adversely affect each other. For example, it may be desirable to further improve an immunosuppressive agent. This type of molecules are suitably present in combination in amounts that are effective for the intended purpose. The active ingredients can also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin microcapsule and poly- (methylmetacylate) microcapsule, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. These types of techniques are described in Remington's Pharmaceutical Sciences, Sixteenth Edition, A. Osal, Ed. (1980). The formulations that are used for in vivo administration must be sterile. This is easily accomplished, for example, by filtration through sterile filtration membranes. Prolonged-release preparations can be provided. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody variant, which matrices are in the form of molded articles, eg, films or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly (2-hydroxyethyl-methacrylate) or poly (vinylalcohol)), polylactides (U.S. Patent No. 3,773,919), L-glutamic acid copolymers and ethyl-L-glutamate, ethylene vinyl acetate not degradable, degradable lactic acid-glycolic acid copolymers, for example, LUPRON DEPOT ™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate) and poly-D- (-) - 3-hydrobutyric acid. While polymers, such as ethylene-vinyl acetate and lactic acid-glycolic acid, are capable of releasing molecules for 100 days, certain hydrogels release proteins for shorter periods of time. When encapsulated antibodies remain in the body for a prolonged period of time, they can denature or form as aggregates of the result of exposure to a humidity of 37 ° C, resulting in a loss of biological activity and possible changes in immunogenicity. You can devise rational strategies for stabilization, depending on the mechanism involved. For example, if it is discovered that the aggregation mechanism is intermolecular S-S formation through the thio-disulfide exchange, stabilization can be achieved by modifying the sulfhydryl residues, by lyophilizing from acid solutions, by controlling the content of moisture, when using appropriate additives and when developing specific polymer matrix compositions. The amount of the therapeutic polypeptide, antibody or fragment thereof, which will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined with standard clinical techniques. When possible, it is desirable to determine the dose-response curve and the pharmaceutical compositions of the invention first in vitro, and then in animal model systems useful prior to testing in humans. In a preferred embodiment, an aqueous solution of the therapeutic polypeptide, antibody or fragment thereof, is administered via subcutaneous injection. Each dose can range from about 0.5 μg to 50 μg per kilogram of body weight, or more preferably, from about 3 μg to 30 μg of kilogram of body weight. The dosing schedule for subcutaneous administration can range from once a month to once a day, which depends on a variety of clinical factors, including the type of disease, the severity of the disease and the sensitivity of the patient. individual to the therapeutic agent.

USES FOR THE ANTIBODY VARIANT The antibody variants of the present invention can be used as affinity purification agents. In this process, antibodies are immobilized on a solid phase, such as, for example, SEPHADEX ™ resin or filter paper, using methods well known in the art. The immobilized antibody variant is contacted with a sample containing the objective to be purified, and subsequently the support is washed with a suitable solvent that will remove practically all the material in the sample, with the exception of the objective to be purified, which is bound to the immobilized antibody variant. Finally, the support is washed with another suitable solvent, such as, for example, glycine buffer, which will release the target of the antibody variant. The variant antibodies can also be used in diagnostic assays, for example, for the detection of expression of an objective of interest in specific cells, tissues or serum. For diagnostic applications, the antibody variant will usually be labeled with a detectable entity. Numerous labels are techniques available to quantify a change in fluorescence and are described below. The chemiluminescent substrate is excited electronically by a chemical reaction and can then emit light that can be measured (using, for example, a chemiluminescence meter) or donate energy to a fluorescent acceptor. Examples of enzymatic labels include luciferases (e.g., firefly luciferase and bacterial luciferase; U.S. Patent No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease, peroxidase, e.g., horseradish peroxidase (HRP), alkaline phosphatase, glucoamylase, lysozyme, saccharide oxidases (for example, glucose oxidase, galactose oxidase and dehydrogenase), heterocyclic oxidases (such as, for example, uricase and xanthine oxidase), lactoperoxidase, microperoxidase, etc. Techniques for conjugating enzymes with antibodies are described by O'Sullivan et al., In the work: Methods for the Preparation of Enzyme-Antibody Conjugates for Use in Enzyme Immunoassay, in Methods in Enzym. (Ed. J. Langone &H. Van Vunakis), Academic press, New York, 73: 147-166 (1981). Sometimes, the label is conjugated indirectly with the antibody variant. The expert technician will know various techniques to achieve this. For example, the antibody variant can be conjugated to biotin, and any of the three categories of labels mentioned above can be conjugated to avidin, or vice versa. Biotin binds selectively with avidin and thereby the label can be conjugated to the antibody variant in this indirect manner. Alternatively, to achieve indirect conjugation of the label with the antibody variant, the antibody variant is conjugated with a small hapten (eg, digloxin) and one of the different types of labels mentioned above is conjugated with an antibody variant. anti-hapten (for example, anti-digloxin antibody). Accordingly, indirect conjugation of the label with the antibody variant can be achieved. In another embodiment of the present invention, the antibody variant does not need to be labeled, and the presence thereof can be detected by using a labeled antibody that binds to the antibody variant. The antibodies of the present invention can be employed in any known assay method, such as, for example, competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays. Zola, Monoclonal Antibodies: A Manual of Techniques, p. 147-158 (CRC Press, Inc. 1987). Competitive binding assays are based on the ability of a labeled standard to compete with the test sample in binding with a limited amount of the antibody variant. The amount of target in the test sample is inversely proportional to the amount of the standard that is linked to the antibodies. To facilitate the determination of the quantity of the standard that is to be linked, antibodies are usually insolubilized before or after competition. As a result, the standard and the test sample that bind with the antibodies can be conveniently separated from the standard and from the test sample that has not yet joined. Sandwich assays involve the use of two antibodies, each capable of binding to a different immunogenic portion, or epitope, or the protein to be detected. In a sandwich assay, the test sample to be analyzed is ligated by a first antibody, which is immobilized on a solid support, and subsequently to a second antibody that binds to the test sample, which forms an insoluble complex of three parts. See, for example, U.S. Pat. 4,376,110. The second antibody itself can be labeled with a detectable entity (direct sandwich assays) or can be measured using an anti-immunoglobulin antibody that is labeled with a detectable entity (indirect sandwich assay). For example, one type of sandwich assay is an ELISA, in which case the detectable entity is an enzyme. For immunohistochemistry, the tumor sample can be cooled or frozen or immersed in paraffin and fixed with a preservative, such as formalin. The antibodies can also be used for in vivo diagnostic assays. Generally, the antibody variant is labeled with a radionucleotide (such as, for example, n? I, 99Tc, 14C, 131I, 3H, 32P or 35S) such that the tumor can be localized using immunoscintigraphy. For example, the high affinity anti-IgE antibody of the present invention can be used to detect the amount of IgE present in, for example, the lungs of an asthmatic patient. The antibody of the present invention can be provided in a kit, that is, a packaged combination of reagents in predetermined amounts with instructions for performing the diagnostic assay. When the antibody variant is labeled with an enzyme, the kit can include necessary substrates and cofactors by means of the enzyme (eg, a substrate precursor that provides the chromophore or detectable fluorophore). In addition, other additives may be included, such as, for example, stabilizers, dampers (for example, a block buffer or lysis buffer), etc. The relative amounts of various reagents can be made to fluctuate widely to provide solution concentrations of reagents that greatly optimize the sensitivity of the assay. In particular, reagents can be provided as dry powders, usually lysed, including excipients, which in the solution will provide a reagent solution having the appropriate concentration.

IN VIVO USES OF THE ANTIBODY It is contemplated that the antibodies of the present invention can be used to treat a mammal. In one embodiment, the antibody is administered, for example, to a non-human mammal for the purposes of obtaining preclinical data. Illustrative non-human mammals to be treated include non-human primates, dogs, cats, radents and other mammals, in which preclinical studies have been carried out. Such mammals can be animal models established for a disease to be treated with the antibody or can be used to study the toxicity of the antibody of interest. In each of these modalities, dose escalation studies in the mammal can be carried out. The antibody or polypeptide is administered by any suitable means, including parenteral, subcutaneous, intraperitoneal, intrapulmonary and intranasal, and, if desired for local immunosuppressive treatment, via intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. In addition, the antibody variant is suitably administered by pulse infusion, in particular with the declining doses of the antibody variant. Preferably, the dosage is given by injections, more preferably, intravenous or subcutaneous injections, depending, in part, on whether the administration is brief or chronic. For the prevention or treatment of diseases, the appropriate dosage of the antibody or polypeptide will depend on the type of disease to be treated, the severity and course of the disease, whether the antibody variant is administered for preventive or therapeutic purposes, prior therapy, the clinical history of the patient and of the response to the antibody variant, and of the agency of the attending physician. The very high affinity human anti-IgE antibodies of the present invention can be appropriately administered to the patient on one occasion or in a series of treatments. Depending on the type and severity of the disease, approximately 0.1 mg / kg to 150 mg / kg (e.g., 0.1-20 mg / kg) of antibody is an initial candidate dose for administration to a patient, either, for example, by one or more separate administrations, or by continuous infusion. A typical daily dosage should fluctuate from approximately 1 mg / kg to 100 mg / kg or greater, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment is prolonged until a desired suppression of the disease symptoms demonstrated. However, other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and trials. An exemplary dosage regimen for an anti-LFA-1 or anti-ICAM-1 antibody is described in WO 94/04188. The antibody variant composition will be formulated, dosed and administered in a manner congruent with good medical practice. Factors to be considered in this context include the particular disorder to be treated, the particular mammal to be treated, the clinical condition of the individual patient, the cause of the disorder, the site of administration of the agent, the method of administration, the administration and administration scheme. Other factors known to medical professionals. The "therapeutically effective amount" of the antibody variant to be administered will be governed by these considerations, and is the minimum amount necessary to prevent, alleviate or treat a disease or disorder. The antibody variant does not need to be formulated, but is optionally made with one or more agents that are currently used to prevent or treat the disorder in question. The effective amount of these other agents depends on the amount of the antibody present in the formulation, the type of disorder or treatment, and other factors discussed above. These are used, in general terms, in the same dosages and with the routes of administration that were used previously or approximately from 1 to 99% of the dosages previously used. The antibodies of the present invention that recognize IgE as their target can be used to treat "IgE-mediated disorders". These include diseases, such as asthma, rhinitis and allergic conjunctivitis (hay fever), eczema, urticaria, atopic dermatitis and food allergies. The serious physiological condition of anaphylactic shock by, for example, bee stings, snakebites, food or medication, is also encompassed within the scope of this invention.

MAPPING THE ANTIBODY EPYTOP The term "epitope" refers to a site on an antigen to which B cells and / or T cells respond. B cell epitopes can be formed from contiguous amino acids or non-contiguous amino acids juxtaposed by the tertiary folding of a protein. Epitopes formed from contiguous amino acids are usually retained on exposure with denaturing solvents, while the epitopes formed meditate tertiary folding are usually lost in the treatment with denaturing solvents. A customary epitope includes at least 3, and more usually, at least 5 or 8 amino acids in a single spatial conformation. Antibodies that recognize the same epitope can be identified in a simple immunoassay that shows the ability of an antibody to block the binding of another antibody to a target antigen. Epitope mapping of the binding site for high affinity IgE antibodies of the present invention involves Western blot analysis for binding, a peptide scan of the CH3 domain of IgE, an alanine scan of the regions showing the binding, the replacement of amino acids from the corresponding regions of IgG1 and site-directed mutagenesis. Peptide screening of the complete IgE CH3 domain requires seventy-three superimposed peptides. Each peptide was subjected to binding by the labeled anti-IgE antibodies of the present invention in order to determine the epitope or specific IgE epitopes that block the binding of IgE with its high affinity receptor. Peptide screening identified two peptides as potential contact sites with anti-IgE MAb, termed Epitope "A" and Epitope "B" (See Figure 11). Although the Epitope "A" and Epitope "B" sequences are approximately 80 amino acids remote in the linear sequence, they are located close to one another in the three-dimensional structure of IgE. Both are exposed surfaces, overlap with the FceRI binding site of IgE, and in both peptides, there are positively charged residues of Arg and Pro hydrophobic residues. Figure 12 illustrates the binding region of Epitope B as determined by ELISA. using the peptide scan. The determination was made on which amino acid residues are critical for the binding of high affinity antibodies within these epitopes by alanine scanning mutagenesis. (Cunningham et al., "High-Resolution Epi Top Mapping of hGH-Receptor Interactions by Alanine-Scanning Mutagenesis" Science 244: 1081-1085). The alanine was substituted for each of the Epitope A and Epitope B residues and the binding of the high affinity monoclonal antibodies was determined. (See Example 12 presented below and Figures 13 and 14).

ACTIVE AND PASSIVE IMMUNIZATION The invention also relates to pharmaceutical compositions, for example, vaccines, which comprise the molecules of the peptide immunogen of the present invention, including SEQ ID NO 72 and / or SEQ ID NO 74, and diluents, excipients, adjuvants or carriers. Furthermore, the present invention relates to a process for the preparation of an immunogen of the present invention, which comprises covalently coupling at least one peptide of the present invention with an entity capable of producing an immune response against the peptide. The present invention also relates to immunogenic peptides as defined above, for use as a pharmaceutical, for example, in the treatment of IgE-mediated diseases or disorders, such as, for example, allergy and atopic dermatitis. The present invention further relates to a method for immunizing a mammal against IgE-mediated diseases or disorders, such as, for example, allergies and atopic dermatitis; the method comprises administering a therapeutically effective amount of immunogenic peptides, as defined above, to a patient in need of this treatment. The immunogenic peptides of the present invention, while practically incapable of mediating the release of non-cytolytic histamine, are capable of producing antibodies with potent serological cross-reactivity with the target amino acid sequences of Epitope A and / or Epitope B. Initial dose of peptide (eg, from about 0.2 mg to 5 mg) can be administered, for example, intramuscularly, followed by repeated doses (booster dose) of the same on days 14 to 28 after. The doses, of course, will depend to some extent on the patient's age, weight and general health. Immunization can be "active" or "passive". In "active" immunization, the individual receives an immunogenic peptide of the present invention and an anti-IgE response is actively induced by the individual's immune system.

The "active" immunization is preferred for use in humans, but other mammalian species can be treated in a similar way, for example, the dog. In this description the term "immunogenic carrier" includes those materials which have the property of independently producing an immunogenic response in a host animal and which can be covalently coupled to the polypeptide either directly via formation or via ester bonds. between the carboxyl, amino or hydroxyl free groups in the polypeptide and the corresponding groups in the immunogenic carrier material, or alternatively by binding through a conventional bifunctional linking group, or as a fusion protein. Examples of this type of immunogenic carriers include: albumins, such as, for example, BSA; globulins; thyroglobulins; homoglobins; hemocyanins (particularly keyhole limpet hemocyanin [KLH, keyhole limpet hemocyanin]; proteins extracted from ascaris, for example, extracts of ascaris such as those described in J. Immunol 111 [1973] 260-268, J. Immunol. 122 [1979] 302-308, J. Immunol 98 [1967] 893-900, Am J. Physiol 199 [1960] 575-578 or purified products thereof, polylysine, lysine-glutamic acid copolymers, copolymers which contain lysine or ornithine, etc. Vaccines have been produced using diphtheria toxoid as an immunogenic carrier material (Lepow ML et al., J. of Infectious Diseases 150 [1984] 402-406; Coen Beuvery, E. et al., Infection and Immunity 40 [1983] 39-45) and these toxoid materials can also be employed in the present invention The purified tuberculin protein derivative (PPD) is particularly preferred for use in the "active" immunization scheme, since (1) ) by itself does not induce a response to T cells (ie, it comes into effect with a "T cell hapten"), and yet behaves like a fully processed antigen and is recognized as such by T cells; (2) it is known to be one of the most powerful "carriers" of hapten in the linked recognition mode; and (3) can be used in humans without further evaluation. The present invention also relates to polynucleotides encoding the peptides of the present invention, vectors comprising these polynucleotides, and cells harboring these vectors. In addition, active immunization can be achieved with the administration of the polynucleotides encoding the peptides of the present invention. Suitable vectors for this type of therapy are commonly used in the art and include, for example, adenovirus vectors. "Passive" immunization is achieved with the administration of anti-IgE antibodies of the present invention to a patient suffering from a disease or IgE-mediated condition. These antibodies can be prepared by administering an immunogenic peptide of the present invention to a non-human mammal and by collecting the resulting antiserum. Improved assessments can be achieved with repeated injections over a period of time. There is no particular limitation with respect to mammalian species, in which the causative antibody can be used; In general terms, it is preferred to use rabbits or guinea pigs, but horses, cats, dogs, goats, pigs, rats, cows, sheep, etc. can also be used. The antibody is recovered by collecting blood from the immunized animal after 1 to 2 weeks after the last administration., by centrifuging the blood and by isolating the serum from the blood. The monoclonal antibodies can be, for example, human or murine. When an individual is immunized, an antibody of the present invention can be introduced into the mammal by, for example, intramuscular injection. However, any form of administration of the antibody can be employed. Any conventional solid or liquid vehicle that is acceptable to the individual and has no adverse side effects can be used. Phosphate-buffered saline (PBS) can be employed at a physiological pH, for example, about pH 6.8 to 7.2, preferably about pH 7.0 as a vehicle, alone or with a suitable adjuvant, such as example, an adjuvant based on aluminum hydroxide. The following examples are offered by way of illustration and in no way as a limitation.

EXAMPLES Example 1 Humanization of Mab TES-C21 murine anti-IgE The sequences of the heavy chain variable region (VH) and the light chain variable region (VL) of Murine TES-C21 mab were compared with human antibody germline sequences available in public databases. Various criteria were used when a mold was decided, as described in step 1 above, among which are included the length, similar CDR position in the framework, overall homology, size of the CDR, etc. All these criteria considered together provide a result for the choice of the optimal human template, as shown in the sequence alignment between the heavy chain and light chain Mab TES-C21 sequences and the respective human template sequences described in the Figures 3A and 3B. In this case, more than one human framework mold was used to design this antibody. The human template chosen for the VH chain was a combination of DP88 (residues 1-95 aa) and JH4b (103-113 residues aa) (See Figure 3B). The human template chosen for the VL chain was a combination of L16 (subgroup III VK, 1-87 residues aa) combined with JK4 (98-107 residues aa) (See Figure 3A). The framework homology between the sequence of murine origin and the human template formulation was approximately 70% for VH and approximately 74% for VL. Once the template was chosen, a Fab library was constructed by DNA synthesis and overlapping PCR, as described above and shown in Figure 2. The library composed of CDRs synthesized from TES-C21 synthesized with the templates respective chosen humans, DP88 / JH4b and L16 / JK4. The complexity of the library was 409Q (= 212). The overlapping nucleotides encoding partial sequences of VH and VL were synthesized in the range of approximately 63 to 76 nucleotides with 18 to 21 overlapping nucleotides. PCR amplification of the VL and VH gene was carried out using a biotinylated forward primer containing the specific sequence, with respect to the framework FR1 region, and a paired sequence paired to the end of the leader sequence (GenelII) and a reverse primer of the conserved constant region (Ck or CH1) under the standard PCR conditions. The PCR product was purified by agarose gel electrophoresis, or by a commercial PCR purification kit in order to remove unincorporated biotinylated primers and by non-specific PCR. The 5'-phosphorylation of the PCR product was carried out using 2 μg of the PCR product, 1 μL of the T4 polynucleotide kinase (10 units / μL), 2 μL of 10X PNK buffer, 1 μL of 10 mM ATP in a total volume of 20 μL adjusted by ddH20. After incubation at 37 ° C for 45 minutes, and thermal denaturation at 65 ° C for 10 minutes, the reaction volume was adjusted to 200 μL by adding ddH20 for the next step. 100 μL of streptavidin-coated magnetic beads were washed twice with 200 μL of 2x B & W buffer and resuspended in 200 μL of 2x B & W buffer. The phosphorylated PCR product was mixed with beads and incubated at room temperature (RT) for 16 minutes with moderate agitation. The beads were pelleted and washed twice with 200 μL of the 2x B & W buffer. The non-biotinylated ssDNA (negative chain) was eluted with 300 μL of fresh 0.15M NaOH prepared at room temperature for 10 minutes with moderate agitation. A second elution with NaOH may slightly increase the yield (optional) The eluent was centrifuged to remove any trace beads. The ssDNA was precipitated from the supernatant by adding 1 μL of glycogen (10 mg / mL), 1/10 volume of 3M NaOAc (pH 5.2) and 2.05 volumes of EtOH. The ssDNA precipitated was then washed with 70% EtOH followed by lyophilization for 3 minutes and the solution in 20 μL of ddH20. The ssDNA was quantified by detecting it on an agarose plate with ethidium bromide (EtBr) with DNA standards, or by measuring the OD260.

Example 2 Cloning of VH and VL in the phage expression vector The VH and VL were cloned into a phage expression vector by mutagenesis and hybridization. Uridinyl templates were prepared by infecting strain CJ236 E. coli (dut ~ ung-) with a phage based on M13 (phage display vector TN003). The following components [200 ng uridinly phage vector (8.49 kb); 92 ng of the H chain of a single phosphorylated strand (489 bases); 100 ng L-chain of a single phosphorylated strand (525 bases); 1 μL of buffer 10X fixative; adjust volume with ddH20 to 10 μL] were fixed (at approximately an 8-fold molar ratio of insert with respect to the vector) by PCR by maintaining the temperature at 85 ° C for 5 minutes (denaturation) and then reducing it to 55 ° C for 1 hour. The samples were cooled on ice. To the fixed product, the following components were added: 1.4μL of 10 X synthesis buffer; 0.5 μL of T4 DNA ligase (1 units / μL); 1 μL of T4 DNA polymerase (1 units / μL) followed by incubation on ice for 5 minutes, and 37 ° C for 1.5 hours. The product was then precipitated with ethanol and dissolved in 10 μL of ddH20 or TE. DNA was digested with 1 μL of Xbal (10 units / μL) for 2 hours, and thermoinactivated at 65 ° C for 20 minutes. The digested DNA was transfected in 50 μL of the electro-component DH10B cells by electroporation. The resulting phage was assessed by growth on XL-lBlue bacterial turf at 27 ° C overnight. The clones were sequenced to confirm the composition.

EXAMPLE 3 Deep well culture for genomic library screening A. Preparation of phage library culture plates The phage library was diluted in LB media to achieve the desired number of halos per plate. High titration phage was mixed with 200 μL of XL-1B cell culture. 3 mL of upper LB agar was mixed, poured into an LB plate and allowed to stand at room temperature for 10 minutes. The plate was incubated overnight at 37 ° C.

B. Phage Elution 100 μL of phage elution buffer "10 mM Tris-Cl, pH 7.5, 10 mM EDTA, 100 M NaCl was added to each well of a sterile 96-well U-well plate. A single halo of phage from the library plate overnight with a pipette filtered into a well.The phage elution plate was incubated at 37 aC for 1 hour.The plate was stored at 4 ° C after incubation.

C. Culturing Probed Well Plates XL1B 50 Ml culture cells were added to the 2xYT media at a 1: 100 dilution. The cells were grown at 37 ° C on a shaker until the Aeoo was between 0.9 to 1.2.

C. Injection with phage in deep well plates When the cells reached the appropriate OD, 1M IPTG (1: 2000) was added to the XL1B culture. The final concentration of IPTG was 0.5 mM. 750 μL of the cell culture was transfected into each well of a 96 well deep well plate (Fisher Scientific). Each well was incubated with 25 μL of eluted phage. The deep well plate was placed on the agitator (250 rpm) and incubated overnight at 37 ° C.

D. Preparation of the supernatant for ELISA screening Following incubation, the deep well plates were centrifuged at 3,250 rpm for 20 minutes using the Beckman JA5.3 plate rotor. 50 μL of supernatant was removed from each well by ELISA.

E. Inoculation of 15 mL of liquid cultures of XL-1 cells XL-1 cells were grown at 37 ° C on the shaker (250 rpm) in 2xYT containing 10 μg / mL tetracycline to A6oo = 0.9 to 1.2. IPTG was added in a final concentration of 0.5 mM and 15 mL of the culture was transfected to a 50 mL conical tube for each clone to be characterized. The cells were inoculated with 10 μL of phage from the high-value raw material (titre = VLO11 milliliter forming units, pfu / mL) and incubated for 1 hour at 37 ° C. The cells were grown overnight at room temperature by applying agitation.

F. Isolation of soluble Fab of periplasm The cells were pelleted in an IEC centrifuge at 4,500 rpm for 20 minutes. The culture medium was removed, the pellets were resuspended in 650 μL of resuspension buffer (50 mM Tris, pH 8.0 containing 1 mM EDTA and 50 mM sucrose), vortexed and placed on ice for 1 hour with light agitation. Cell debris was removed by centrifugation at 9,000 rpm for 10 minutes at 4 ° C. The supernatant containing the soluble Fabs was collected and stored at 4 ° C.

Example 4 Modification of the framework There were twelve murine / human diffuse residues in the framework in the potential key positions described above. Position 73 in VH remained as the murine threonine residue in the humanization library, because it was determined that this position affects the binding. However, it was perceived that threonine in VH 73 is a common human residue in subgroup 1 and 2 of VH of the human germ line. The framework residues that differed between the TES-C21 sequence and the human template were replaced randomly, as described above, and then a potential effect on the binding of the target and on the folding of the antibody was evaluated. Potential framework residues that could affect the union were identified. In this case, these were residues 12, 27, 43, 48, 67, 69 in VH, and 1, 3, 4, 49, 60, 85 in VL (Kabat numerical system). (See Figure 4). It was later shown that only positions 27 and 69 significantly affected the binding in the VH region (clone number 1136-2C). The primary screening employed was single-point ELISA (SPE) analysis using culture media (see the following description). Primary screening selected clones that bind to the target antibody molecule. Clones that provided an equal or better signal than the progenitor molecule were selected for the next screening cycle. In the second screening cycle, individual phages were grown in 15 ml of bacterial culture and periplasmic preparations were used for the SPE and ELISA assays. Clones that retained a superior bond in this assay were further characterized. Once all the selected primary clones were processed, 10-15% of clones were sequenced and the clones were configured according to the sequence. Representatives of each sequence group were compared with others and the best clones were selected. The sequences of these chosen clones were combined and the effects of various combinations were evaluated. The constructed library was screened for ELISA to improve its binding to recombinant human IgE. Clones with higher binding affinity than that of murine Fab TES-C21 were isolated and sequenced. Clone ID # 4, 49, 72, 76 and 136 were further characterized. The ELISA titration curves for clone 4, 49, 72, 78 and 136 are shown in Figures 5A and 5B indicating that their affinity is similar to the progenitor, TES-C21. These clones compete with murine TES-C21 to bind to human IgE, indicating that the binding epitope did not change during the humanization process. Humanized Fabs did not bind to IgE linked to FceRI, suggesting that humanized antibodies are less likely to cross-link the receptor to elicit histamine release when they are constructed on divalent IgG. Humanized clone 136 retained 5 murine heavy chain framework residues (= 94.3% human VH framework homology), with a 100% human light chain lattice selected by affinity maturation. The inhibition of IgE binding with FceRI was demonstrated by humanized Fab (Figure 6).

Example 5 Single-point ELISA protocol for anti-IgE screening Plates were coated with 2ug / mL of bovine anti-human Fd in carbonate coating buffer overnight at 4 ° C. The coating solution was removed and the plates were blocked with 200 uL / well of 3% BSA / PBS for 1 hour at 37 ° C. After washing the plates 4 times with PBS / TWEEN® 0.1% (PBST), 50 uL / well of the Fab sample was added (ie, supernatant containing high titration phage and secreted Fab or periplasmic prep from the DMB block, or 15 mL of prep). Plates were incubated for 1 hour at room temperature followed by washing four times with PBST. Then, 50 uL / well of biotinylated SE44 at 0.015 ug / Ml diluted in 0.5% BSA / PBS and 0.05% TWEEN® were added. The plates were then incubated for 2 hours at room temperature and washed four times with PBST. 50 uL / well of a 1: 2000 dilution of StreptAvindin HRP in 0.5% BSA / PBS and 0.05% TWEEN® were added and the plates were incubated 1 hour at room temperature. The plates were washed six times with PBST. 50 μL / well of TMB substrate (Sigma) was added to develop and then stopped by adding 50uL / well of H2S04 0.2M.

EXAMPLE 6 ELISA Assay: Anti-IgE Plates were coated with 0.25 μg / mL (for ug / mL of purified Fab) of SE44 in a carbonate coating buffer overnight at 4 ° C. The coating solution was removed and the plates were blocked with uL / well of 3% BSA / PBS for 1 hour at 37 ° C. Plates were washed four times with PBS / 0.1T TWEEN® (PBST). 50 μL / well of Fab (from 15 mL of prep periplasmic) was added starting with a dilution of 1: 2 and diluting 3 times serially in 0.5% BSA / PBS and 0.05% TWEEN®. Plates were incubated for 2 hours at room temperature. Plates were washed four times with PBST and 50 uL / well of a 1: 1000 dilution (0.8 ug / ml) of sheep anti-human Fd biotin in 0.5% BSA / PBS was added and 0.05% TWEEN® was added. 20. The plates were incubated again for two hours at room temperature. After a quadruple wash with PBST, 50 uL / well • of Neutra-avidin-AP 1: 2000 (0.9 ug / ml) in 0.5% BSA / PBS and 0.05% TWEEN® 20 were added and the plates were incubated for 1 hour at room temperature. Plates were washed four times with PBST and developed by adding 50 uL / well pNPP substrate. The development was stopped by adding 50 uL / well of 3M NaOH. The absorbance of each well was read at 405 nm or 410 nm.

Example 7 Protocol for affinity purification of soluble Fab expressed in M13 phage A. DAY 1 Two cultures of 500 mL (2xYT) containing 100 mg / mL of tetracycline were inoculated with 5 mL of XL1B of raw material overnight and allowed to grow at 37 ° C to A600 = 0.9 to 1.2. IPTG was added at a concentration of 0.5 mM. The cell culture was then infected with 200 μl of phage per culture and incubated for 1 hour at 37 ° C with shaking. After infection, the cells were grown at 25 ° C overnight with shaking.

B. DAY 2 The cells were pelleted at 3500 x g for 30 minutes at 4 ° C in 250 L centrifuge tubes. The culture medium was aspirated and the pellets resuspended in a total volume of 12-15 mL of buffer lysis (Shock absorber A + protease inhibitor cocktail).

Shock absorber A: (1 liter) 50 mM NaH2P04 6.9 g of NaH2P04H20 (or 6 g of 300 mM NaCl NaCl 17.54 g of Imidazole NaCl 10 mM 0.68 g of imidazole (MW 68.08) Adjust pH to 8.0 using NaOH Shock absorber lysis: Mix 25 mL of Shock absorber A with a cocktail tablet of the complete protease inhibitor (Roche, Basel, Switzerland) The resuspended cells were transfected to a 50 mL conical tube and lysed with 100 μL 100 mg / mL of lysozyme by inverting the tube several times until the mixture moves together as a uniform mass (due to lysis). The cells were sonicated on ice followed by the addition of 10 μL of DNase I (approximately 1000 units) and shaken carefully at 4 ° C for 30 minutes. The wastes were pelleted by centrifugation at 12,000 x g for 30 minutes at 4 ° C, using 50 mL centrifuge tubes. The supernatants were transfected into a new conical tube and stored at 4 ° C. Ni-NT agarose (Qiagen, Valencis, CA) was used to purify the soluble Fabs according to the manufacturer's protocol. The lysate was mixed with Ni-NTA t and loaded onto a column. The through flow was collected for the SDS-PAGE analysis. The column was washed with 20 mL of buffer (50 mM NaH2P04, 300 mM NaCl, 15 mM imidazole, adjust pH to 8.0 with NaOH) followed by a 20 mL wash with 50 M NaH2P04, 300 mM NaCl, 20 mM imidazole. The Fabs were eluted with 6 x 500 μL of elution buffer (50 mM NaH2P04, 300 mM NaCl, 450 mM imidazole, adjusted pH to 8.0 with NaOH) and analyzed by SDS PAGE and the fraction with the largest amount and dialyzed was selected in PBS at 4 ° C.

Example 8 Soluble receptor assay A 96-well plate suitable for ELISA analysis was coated with 0.05 mL 0.5 μg / mL of alpha chain FceRI receptor coating buffer (50 mM carbonate / bicarbonate, pH 9.6) for 12 hours at 4 hours. -8 ° C. Wells were aspirated and 250 μL of blocking buffer (PBS) was added, 1% BSA, pH 7.2) and incubated for 1 hour at 37 ° C. In a separate assay plate, reference samples and MAB TES-C21 were titrated from 200 to 0.001 μg / mL by 1: 4 dilutions with a test buffer (0.5% BSA and 0.05% TWEEN® 20, PBS, pH 7.2) and an equal volume of 100 ng / mL of biotinylated IgE was added and the plate was incubated for 2-3 hours at 25 ° C. The wells coated with FceRI were washed three times with PBS and 0.05% TWEEN® 20, and 50 μL of the sample wells were transfected and incubated with shaking for 30 minutes at 25 ° C. 50 μL / well of 1 mg / mL streptavidin-HRP, diluted 1: 2000 in a test buffer, were incubated for 30 minutes with shaking and then the plate was washed as before. 50 uL / well of TMB were added and color developed. The reaction was stopped by adding an equal volume of 0.2M H2SO4 and measured by the absorbance at 450 nm.

EXAMPLE 9 Binding of antibodies with FceRI loaded with IgE The binding of antibody with human IgE associated with the alpha subunit of FceRI was determined with preincubation with 10 μg / mL of human IgE for 30 minutes at 4 ° C. Plates were washed three times followed by 1 hour of incubation with various concentrations of either MAb E-10-10 anti-human IgE or with the humanized Fab variant. The binding of the Fabs was detected with an anti-human Fd antibody labeled with biotin followed by SA-HRP. Murine E-10-10 MAb was detected by Ab conjugated with goat anti-mouse IgP HRC HRc.

Example 10 Characterization of clones Each candidate was assessed for binding affinity and positive clones were sequenced. Antibody variants that had beneficial mutations in the CDR regions that increase the binding affinity were further characterized. The trials included Biacore analysis: inhibition of IgE binding to its receptor; and crosslinking of IgE bound to the receptor. A library of variants was created. The amino acid sequences for the various CDRs that showed better affinity are described in Table 1. Figure 7 presents high affinity candidates having combinations of substitutions. TABLE 1 Progenitor Figure 19 shows 19 heavy chain variants and 35 light chain variants are presented in Figure 8. Three candidates were further characterized to evaluate their binding affinity and these are presented in Table 2. TABLE 2 Affinity binding Example 11 Expression and purification of anti-IgE antibodies and conjugation by HRP Candidate high affinity MAbs were generated. For the generation of intact anti-IgE MAbs, the light chain and heavy chain variable regions were amplified by PCR from phage vector templates and subcloned separately into H and L chain expression vectors under the expression of a promoter. CMV. Six complete antibody clones were constructed and are depicted in Figures 10A-F. Suitable light chain and heavy chain plasmids were co-transfected into the mouse myeloma NSO cell line using electroporation by techniques commonly used in the art. See, for example, Liuo et al., J. Immunol. 143 (12): 3967-75 (1989). The antibodies were purified from the supernatants of the single stable cell line using protein A-sepharose (Pharmacia). The concentration of the antibody was determined using a spectrophotometer at 280 nm and an FCA assay (IDEXX). The purified antibodies were conjugated by horseradish peroxidase (HRP) - using the peroxidase conjugation kit (Zymed Labs, San Francisco, CA) according to the manufacturer's protocol. The titration of each conjugated anti-IgE MAb was determined using ELISA with plates coated with a monoclonal human IgE (SE44). The following crops have been deposited with American Type Culture Collection, 10801 University Boulevard, 20110-2209 USA. (ATCC): This deposit was made with the stipulations of the Budapest Treaty in the International Recognition of Deposit of Microorganisms for the Purpose of Patent Procedure and the Regulations established therein (Budapest Treaty). This ensures the conservation of a viable crop for 30 years from the date of deposit. The agency will be available through the ATCC under the terms of the Budapest Treaty, which ensures the permanent and unrestricted availability of offspring from the crop to the public with the issuance of the relevant United States patent. The assignee of the present application has agreed that if the crop on deposit died, lost or destroyed at the time of being under cultivation under suitable conditions, it will replace it immediately, upon notification, with a viable specimen of the same crop. The availability of the deposited strain does not constitute the authorization to practice the invention in contravention of the rights granted in accordance with the authority of any government in accordance with its patent laws. It is considered that the above written specification is sufficient to enable one skilled in the art to practice the invention. The present invention is not limited to the scope of the cultures deposited, since the deposited embodiments are for illustrative purposes for one aspect of the invention, and any culture that is functionally equivalent is considered within the scope of this invention. The deposit of the material mentioned in this document does not constitute an admission that the written description contained in this document is inadequate to enable the practice of any aspect of the invention, including the best way to carry it out, nor does it constitute a limiting the scope of the claims to the specific illustration that this represents. In fact, from the above description, for those skilled in the art, various modifications of the invention will be apparent, in addition to those shown and described herein, these are within the scope of the appended claims.

Example 12 Mapping of high affinity binding epitope of human IgE A. Peptide synthesis and anti-IgE binding assay Some studies have shown that IgE binds to its receptor through the CH3 domain. Since the anti-IgE HA antibodies of the present invention very efficiently block IgE binding to its receptor, we mapped the epitope using peptides spanning the entire CH3 domain. First, we prepare two V5-tagged peptides, one consisting of the entire constant region of human IgE and one consisting of only the CH2-CH3 region of human IgE. These two peptides were expressed by in vitro transcription-translation and used in the Western blot assay to detect anti-IgE Ha MAb binding. Both Mab CL-2C and CL-5I were able to bind intact human IgE as well as to both peptides. To map the epitope with greater specificity, the 73 superimposed peptides comprising amino acid residues 141 to 368 of human IgE, which include the complete CH3 domain, were synthesized. Each peptide is constituted by 12 amino acid residues having 3 amino acids superimposed with the 3 'end of the previous peptide. The SPOT membranes were synthesized with fluoreniimethoxycarbonyl amino acids (Fmoc) in a cell membrane. The membranes were rinsed in methanol and then washed in TBS (ris-bu ffered saline, buffered with Tris) (pH 7.5) three times for 10 minutes. After an overnight incubation in blocking solution (5% milk or 3% BSA in TBS), HRP-tagged Anti-IgE MAbs diluted in blocking solution were incubated with the membrane for 3 hours. After washing three times for 15 minutes in TBS-TWEEN®, using SuperSignal HRP substrate (Pierce), IgE reactivity was measured by exposure to chemiluminescence of BioMax MS film (Kodak) for the desired time. The results obtained by the experiment indicate that Anti-IgE HA MAbs bind to two regions in the CH3 portion of IgE, which are represented by the following two peptide sequences: NPRGVSAYLSRP (epitope "A") and HPHLPRALMRST (Epitope "B") "). (See Figure 12). The union with Epitope A was several times weaker than with Epitope B.

B. Alanine scan mutagenesis An alanine scan was carried out with the amino acid substitutions in the peptides with those found in IgGl in order to determine which amino acids are critically involved in the binding of the Anti-IgE HA MAb with these peptides. The amino acids that were determined to be important for MAb Anti-IgE HA binding were replaced using an in vitro mutagenesis strategy in the IgE e chain. In this study, another peptide covering the Ce2 and Ce3 regions was also used, as described above. (See Figures 13 and 14). The EU numerical scheme for human IgE amino acid residues has been used. The polymerase chain reaction (PCR) to amplify the complete Fc region of IgE, and a truncated form of Fc IgE containing only the CH2-CH3 domain. The DNA products were cloned directly into the pcDNA3 expression vector (Invitrogene, Carisbad, CA) using TOPO cloning (Invitrogene, Carisbad, CA). Mutagenesis was carried out in Fc IgE using superposition PCR (Ho et al., 1989). The DNA products were purified by agarose gel electrophoresis, digested with one or more suitable restriction enzymes, and subcloned into the pcDNA3 expression vector. For each variant construct, the amplified regions were completely sequenced by PCR using the dideoxynucleotide sequencing method from the DNA strands. The recombinant human IgE Fc and its mutants were expressed using a coupled in vitro transcription and translation system based on reticulocyte lysate (Promega, Madison, Wl). The lysates of this coupled in vitro transcription and translation system (10 μl of reaction mixture) were subjected to SDS-PAGE (12%) and then transfected into nitrocellulose membranes. The membranes were blocked with 5% milk powder in saline buffered with Tris (TBS) and subsequently stained with the primary antibody., the Anti-LGE MAb. Specific reactive bands were harvested using a horseradish peroxidase-conjugated goat anti-human IgG Fc (Jackson Labs, Bar Harbor, Maine) and immunoreactive bands were visualized using the Western SuperSignal transfer detection kit (Pierce). The anti-V5 antibodies were used as a positive control that detected the V5 tag introduced at the C-terminus of these peptides. Western blotting with anti-V5 antibodies showed that all peptides were expressed at almost the same level. Interestingly, the anti-IgE Ha MAb were able to bind to the peptide carrying mutations in the Epitope "A", but, they did not bind to the peptide carrying mutations in the Epitope "B", indicating that this second site was more important for the union. (See Figure 15).

EXAMPLE 13 Active Immunization of Transgenic Mice Using an Immunogenic Epitope B Peptide Transgenic mice were used that constitutively expressed human IgE in order to demonstrate the active production of antibodies with a human immunogenic peptide of Epitope B. Two fusion peptides were chemically synthesized, each comprising an immunogenic peptide of the invention, a residue of cysteine and KLH. The sequence of peptide 1 was: (KLH-Cys) - Leu Pro Arg Ala Leu Met Arg Ser Thr and the sequence of peptide 2 was: Leu Pro Arg Ala Leu Met Arg Ser Thr - (Cys-KLH) Transgenic mice were injected subcutaneously with 20 μg of immunogenic peptide in a complete Freud's adjuvant (Difco Laboratories, Detroit, MI) in 200 μL of PBS pH 7.4. At two-week intervals the mice were injected twice subcutaneously with 20 μg of the immunogenic peptide in a complete Freud's adjuvant. Then, two weeks later and three days before sacrificing them, again the mice were injected intraperitoneally with 20 μg of the same immunogen in PBS. Serum was collected and evaluated for the presence of anti-IgE antibodies specific for Epitope B. As seen in Figure 16, the peptide was fixed on the anti-IgE antibodies in these transgenic mice. Those skilled in the art will recognize, or be able to ascertain using only routine experimentation, many equivalents of the specific embodiments of the invention described in this disclosure. It is intended that the following claims encompass these equivalents.

Claims (33)

1. CLAIMS: 1. An isolated peptide, suitable for use as an anti-IgE epitope, comprising the amino acid sequence: Asn Pro Arg Gly Val Ser Xaa Tyr Xaa Xaa Arg Xaa. The peptide according to claim 1, wherein the amino acid sequence is: Asn Pro Arg Gly Val Ser Ala Tyr Leu Ser Arg Pro. 3. An isolated peptide, suitable for use as an anti-IgE epitype, comprising the sequence of amino acids: Leu Pro Arg Ala Leu Xaa Arg Ser Xaa. 4. The peptide according to claim 3, wherein the amino acid sequence is: a) Leu Pro Arg Ala Leu Met Arg Ser Thr; b) His Pro His Leu Pro Arg Ala Leu Met Arg Ser Thr; or c) Leu Pro Arg Ala Leu Met Arg Ser Thr Thr Lys Thr. 5. A composition comprising a peptide according to any of claims 1 to 4 and a physiologically acceptable carrier, diluent, stabilizer and / or excipient. 6. The composition according to claim 5, further comprising an immunogenic carrier. 7. The composition according to claim 6, wherein the immunogenic carrier is selected from the group consisting of BSA, KLH, tetanus toxoid and diphtheria toxoid. The peptide according to any of claims 1 to 4 fused with an immunogenic sequence. The peptide according to claim 8, wherein the immunogenic sequence comprises BSA, KLH, tetanus toxoid and diphtheria toxoid. A composition for inducing an immune response to IgE in a mammal, comprising the peptide or composition according to any of claims 1 to 9, in an amount sufficient to induce a response in the mammal 11. An isolated antibody that specific antibody binds to the peptide according to any of claims 1 to 4. 12. An isolated antibody that specifically binds to the peptide according to any of claims 1 to 4 with similar binding affinity to IgE as antibodies CL-5A, CL- 2C or CL5I that can be obtained by culturing CL-5A Anti-IgE (ATCC No: PTA-5679), CL-2C Anti-IgE (ATCC No: PTA-5678) or CL-5I Anti-IgE (ATCC No: PTA- 5680), respectively. 13. An isolated antibody according to claim 12, said antibody is CL-5A, CL-2C or CL-5I which can be obtained by culturing CL-5A Anti-IgE (ATCC No: PTA-5679), CL-2C Anti-IgE (ATCC No: PTA-5678) or CL-5I Anti-IgE (ATCC No: PTA-5680) hybridoma, respectively. The antibody according to any of claims 11 to 13, further comprising a label. 15. The antibody according to any of claims 11 to 14, wherein the antibody is: a) a chimeric antibody; b) a single chain antibody; c) a Fab fragment; d) an F (ab ') fragment; e) a human antibody; or f) a humanized antibody. 16. A composition comprising an antibody according to any of claims 11 to 15 and a physiologically acceptable carrier, diluent, stabilizer and / or excipient. 17. A method for preparing an antibody according to any of claims 11 to 15; the method comprises: a) immunizing a non-human animal with a peptide according to any of claims 1 to 9 under conditions to produce a response to the antibody, b) isolating antibodies from the non-human animal, ec) identifying an antibody that is specifically binds with high affinity to the peptide according to any of claims 1 to 4. 18. An antibody that can be obtained by a method according to claim 17. 19. A composition comprising the antibody according to claim 18 and a carrier, diluent, stabilizer and / or physiologically acceptable excipient. The antibody according to claim 11 or claim 12, comprising a light chain having at least one sequence selected from SEQ ID No. 5 to 13. The antibody according to claim 11 or claim 12, comprising a chain Weighing having at least one sequence selected from SEQ ID No. 14 to 29. 22. The antibody according to claim 11 or claim 12, comprising a light chain and a heavy chain having at least one sequence selected from SEQ ID No. 5 to 29. 23. An antibody according to claim 22, comprising SEQ ID No. 5, 8, 13, 15, 18 and 26. 24. A method for making a monoclonal antibody, the method comprising: a) immunizing a non-human animal with a peptide according to any of claims 1 to 9, under conditions to produce a response to the antibody, b) isolating from the animal not human the cells that produce the antibody, c) fuse cells that produce the antibody with immortalized cells to form hybridoma cells that produce monoclonal antibody, d) cultivate the hybridoma cells, and e) isolate a monoclonal antibody from the culture that is specifically one to a peptide according to any of claims 1 to 4. 25. A monoclonal antibody obtainable by a method according to claim 24. 26. A composition that comprises of the monoclonal antibody according to claim 25 and a physiologically acceptable carrier, diluent, stabilizer and / or excipient. 27. The antibody according to claims 11 to 15, wherein the antibody is isolated by screening an expression library Fab. 28. The antibody according to claims 11 to 15, wherein the antibody is isolated by screening a combinatorial immunoglobulin library. 29. A kit comprising the antibody according to any of claims 11 to 28. 30. The use of an antibody according to any of claims 11 to 28 for the manufacture of a medicament for use in the treatment of a mammal suffering from, or is at risk of developing a disease or condition mediated by IgE. 31. The use of an antibody generated using a peptide or composition according to any of claims 1 to 9 for the manufacture of a medicament for use in the treatment of a mammal suffering from or at risk of developing an IgE-mediated disease or condition. . 32. The use according to claim 30 or 31, wherein the disease or condition mediated by IgE is allergy, asthma, allergic rhinitis, atopic dermatitis, urticaria or eczema. 33. The use according to any of claims 30 to 32, wherein the mammal is a human.