KR101581659B1 - IDENTIFICATION OF NOVEL IgE EPITOPES - Google Patents

Abstract

The present invention relates to novel peptide epitopes derived from the CH3 domain of IgE that are recognized by high affinity antibodies that specifically bind IgE. These novel peptides include active immunization of individuals that produce high affinity antibodies in an individual and the ability of an individual to generate high affinity anti-IgE antibodies in non-human hosts that specifically bind to these regions of IgE Can be used for passive immunization.

Description

Identification of new IｇE epitopes {IDENTIFICATION OF NOVEL IgE EPITOPES}

The present invention relates to a novel peptide epitope 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 active immunization in mammals producing high affinity antibodies in mammals by administering these peptides. Peptide epitopes can also be used to produce high affinity anti-IgE antibodies that specifically bind to these regions of IgE in non-human hosts, which produced antibodies are used for passive immunization of mammals.

Allergy is a hypersensitivity condition induced by an overactive immune response to foreign substances, such as an allergen. Immediate hypersensitivity, characterized as an allergic reaction immediately after contact with the allergen, is mediated through B cells and is based on an antigen-antibody response. Delayed hypersensitivity is mediated through T cells and is based on the mechanism of cellular immunity. Recently, "allergy" has been almost considered synonymous with type I hypersensitivity reactions.

Immediate hypersensitivity reaction is a reaction based on the production of immunoglobulin class E antibodies (IgE antibodies) by B cells that differentiate into antibody-secreting plasma cells immediately after exposure to an allergen. IgE-induced reactions are localized events that occur at the site of body invasion of the allergen, that is, the mucosal surface and/or local lymph nodes. Locally produced IgE first sensitizes local mast cells, in other words, the IgE antibody binds to the Fcε receptor as a constant region on the surface of the mast cell, and then “spill over” IgE. Enters the circulatory system and binds to receptors in both basophils and tissue-fixed mast cells that circulate throughout the body. When bound IgE subsequently comes into contact with an allergen, the Fcε receptor is cross-linked by the binding of the allergen, which causes cells to degranulate and release a number of anaphylaxis mediators such as histamine, prostaglandin, leukotriene, etc. Are combined. The release of these substances is a typical clinical symptom of immediate hypersensitivity reactions, ie smooth muscle contraction in the respiratory or intestines; Dilation of small blood vessels and increased permeability of them to water and plasma proteins; For example, allergic rhinitis, atopic eczema, secretion of mucus causing asthma; It causes itchiness and pain in the skin, causing irritation of the nerve ends. In addition, after the second contact with the allergen, the reaction is intensified, because some B cells express IgE on the cell surface after the first contact with the allergen, resulting in the “memory pool (sigE+ B cells) of surface IgE-positive B cells (sigE+ B cells). memory pool)”.

There are two major receptors for IgE: high affinity receptor FcεRI and low affinity receptor FcεRII. FcεRI is mainly expressed on the surface of mast cells and basophils, but low levels of FcεRI can also be found in human Langerhan's cells, dendritic cells, and monocytes, where it is IgE-mediated. It functions in the presentation of allergens. In addition, FcεRI has been reported in human eosinophils and platelets (Hasegawa, S. et. al., Hematopoiesis, 1999, 93:2543-2551). FcεRI is not found on the surface of B cells, T cells, or neutrophils. Expression of FcεRI in Langerhans cells and cutaneous dendritic cells is functionally and biologically important for the presentation of IgE-bound antigens in allergic individuals (Klubal R. et al., J. Invest. Dermatol. 1997, 108(3): 336-42).

The low affinity receptor, FcεRII (CD23), is a lecithin-like molecule whose head structure contains three identical subunits extending from a long α-helix twisted stem from the cellular plasma membrane (Dierks, AE et al., J. Immunol. 1993, 150:2372-2382). After binding to IgE, FcεRII binds to CD21 in 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. al., Eur. Resp. J. 1996, 9:63s-66s). FcεRII has long been recognized as being involved in the presentation of allergens (Sutton and Gould 1993, Nature, 366:421-428). IgE bound to FcεRII in epithelial cells leads to specific and rapid allergen presentation (Yang, P.P., J. Clin. Invest., 2000, 106:879-886). FcεRII is present in several cell types, including B cells, eosinophils, platelets, natural killer cells, T-cells, vesicle dendritic cells, and Langerhans cells.

Structural parts have also been identified in IgE molecules that interact with FcεRI and FcεRII. In a mutagenesis study, the CH3 domain of IgE is FcεRI (Presta et al., J. Biol. Chem. 1994, 269:26368-26373; Henry AJ et al., Biochemistry, 1997, 36:15568-15578) and FcεRII ( Sutton and Gould, Nature, 1993, 366: 421-428; Shi, J. et al., Biochemistry, 1997, 36:21 12-2122). The binding sites for both high and low affinity receptors are located symmetrically along the central axis of rotation through the two CH3 domains. The FcεRI-binding site is located in the CH3 domain outwardly around the junction of the CH2 domain, while the FcεRII-binding site is located at the carboxy terminus of CH3.

A promising concept for the treatment of allergies is the use of monoclonal antibodies, which are IgE isotype-specific and are capable of binding to IgE. This approach is based on the inhibition of allergic reactions by downregulating the IgE immune response, which is the first phenomenon in the induction of allergies and provides maintenance of the allergic state. Since the reactions of other antibody types are not affected, immediate and long-term effects on allergic symptoms are achieved. In an early study of human basophil density, a correlation was found between IgE levels in patient plasma and the total number of FcεRI receptors per basophil (Malveaux et al., J. Clin. Invest., 1978, 62:176). In allergic and non-allergic individuals, the FcεRI density was found to be between 10 ^{4 and 10 6 receptors per basophil.} Subsequently, treatment of allergic diseases with anti-IgE was found to reduce the amount of circulating IgE to 1% of the pretreatment level (MacGlashan et al., J. Immunol., 1997, 158:1438-1445). MacGlashan analyzed sera obtained from patients treated with total anti-IgE antibodies that bind to free IgE circulating in patient serum. It has been found that lowering the level of circulating IgE in patients reduces the total number of receptors on the surface of basophils. Thus, it was hypothesized that the FcεRI density at the surface of basophils and mast cells is directly or indirectly regulated by the level of circulating IgE antibodies.

More recently, WO 99/62550 describes the usefulness of IgE molecules and fragments that block IgE binding to receptors by binding to FcεRI and FcεRII IgE binding sites. However, effective treatments in the management of these allergic diseases in the absence of harmful side effects are limited. Another treatment modality for treating allergic diseases is the use of humanized anti-IgE antibodies to treat allergic rhinitis and asthma (Corne, J. et al., J. Clin. Invest. 1997, 99:879-887; Racine Poon, A. et al., Clin. Pharmcol. Ther. 1997, 62:675-690; Fahy, JV et al., Am. J. Resp. Grit. Care Med. 1997, 155:1824-1834; Boulet, LP et al. 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 demonstrate that inhibition of the binding of IgE to its receptors is an effective way to treat allergic diseases.

Antibodies suitable as anti-allergic sources should be able to react with surface IgE positive B cells that differentiate into IgE producing plasma cells and be used to functionally eliminate these B cells. However, in principle, antibodies to IgE can also induce the release of intermediates from IgE-sensitized mast cells by cross-linking the Fcε receptors to antagonize the beneficial effects exerted at the serum IgE and sigE+ B cell levels. One of the potentially harmful problems in the process of developing anti-IgE therapeutics is the possibility of IgE-crosslinking caused by binding of therapeutic antibodies to IgE already bound to high affinity receptors and inducing histamine release leading to a potential anaphylactic response. to be.

For this reason, antibodies used in the treatment of allergies must be able to maintain the ability to recognize sigE+ B cells without interacting with sensitized mast cells and IgE bound to basophils. Such IgE isotype-specific antibodies are, for example, Chang et al., Biotechnology 8, 122-126 (1990), European Patent No. EP0407392, several U.S. It is described in a patent (for example, U.S. Patent No. 5,449,760).

Peptides used to produce anti-IgE antibodies are also not free from the potential risk of inducing anaphylactic antibodies. If antibodies produced during immunization bind to high affinity IgE receptors or IgE bound to other structures, the production of anti-IgE antibodies during active vaccination is histamine release in the same manner as passively administered anti-IgE antibodies. Can attract.

Therefore, there is a need for a peptide for active immunity that specifically binds to IgE but does not induce higher affinity non-anaphylactic antibodies and anaphylactic antibodies that do not bind to IgE already bound to high affinity receptors. The present inventors have identified a specific epitope of IgE that provides high affinity binding of the antibody without binding to IgE in mast cells or basophils. These specific epitopes can be used to produce specific peptides for active immunity to produce antibodies against IgE that bind only to the region of IgE that binds to the receptor, and these antibodies do not cross-link IgE already bound to the receptor. So that anaphylaxis does not occur.

One immunogen of the present invention (Epitope A, Figure 11) comprises the following amino acid sequence:

Asn Pro Arg Gly Val Ser Xaa Tyr Xaa Xaa Arg Xaa (SEQ ID NO. 72).

One example of epitope A is:

Asn Pro Arg Gly Val Ser Ala Tyr Leu Ser Arg Pro (SEQ ID NO. 73).

Another immunogen (Epitope B. Figure 11) contains the following amino acid sequence:

Leu Pro Arg Ala Leu Xaa Arg Ser Xaa (SEQ ID NO. 74).

An example of epitope B is:

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 SEQ ID NO: 72 or SEQ ID NO: 74, Xaa is any amino acid.

These peptides may be included in compositions containing at least one peptide and a physiologically acceptable carrier, diluent, stabilizer or excipient as well as an immunological carrier. Immunological carriers are, for example, BSA, KLH, tetanus toxoid, diphtheria toxoid. In addition, the present invention is SEQ ID NO. It relates to a polynucleotide encoding 72-77, a vector containing the polynucleotide, and a cell containing the vector.

Further, the present invention relates to an antibody that specifically binds to epitope A and/or epitope B. Further, the present invention relates to a method for producing an antibody that specifically binds to epitope A and/or epitope B.

In addition, the present invention relates to administration of a peptide comprising SEQ ID NO: 72 and/or SEQ ID NO 74 to an individual suffering from an IgE-mediated disease or abnormality.

In addition, the present invention relates to administration of a high affinity antibody produced using a peptide comprising SEQ ID NO: 72 and/or SEQ ID NO 74 to a mammal suffering from an IgE-mediated disease or abnormality. High affinity antibodies are human, humanized or chimeric antibodies. Antibodies are polyclonal or monoclonal antibodies. IgE-mediated diseases or diseases include, for example, asthma, atopic dermatitis, urticaria, allergic rhinitis, and eczema.

As shown in Fig. 16, the peptide according to the present invention induced anti-IgE antibodies in these transgenic mice.

1 is a schematic diagram of a phage vector used for antibody cloning and screening.
2 is a schematic diagram of oligonucleotides useful for producing antibody variants.
3A shows a comparison of the light chain of the murine anti-IgE antibodies TES-C21 and the associated human template of L16 and JK4.
3B shows a comparison of the heavy chain of JH4b with TES-C21 and associated human template DP88.
Figure 4 presents a table of framework residue variants with high affinity compared to the parental TES-C21.
5A and B show ELISA titration curves for clones 4, 49, 72, 78, and 136 compared to the parental Fab and negative control (5D12) of TES-C21.
6 shows the results of inhibition tests of clones 2C, 5A, and 5I compared to parental TES-C21 and negative control antibodies.
Figure 7A depicts the sequence of clones carrying a combination of beneficial mutations leading to higher affinity for IgE.
Figures 8A & 8B show the framework sequences of the entire light chain variable region for clones 136, 1, 2, 4, 8, 13, 15, 21, 3O, 31, 35, 43, 44, 53, 81, 9O, 113. do.
Figures 9A & 9B show the framework sequences of the entire heavy chain variable region for 35 clones.
Figure 10 AF shows the complete heavy and light chain sequences for clones 136, 2C, 5I, 5A, 2B, and 1136-2C.
In Fig. 11, the amino acid sequence of the CH3 region of human IgE is shown, and epitope "A" and epitope "B" are emphasized.
12 shows the overlapping peptides used to identify epitope B.
13 shows the identity of the important residue in the binding region of epitope A.
14 shows the identity of the important residue in the binding region of epitope B.
15 shows the results of Western blot analysis of MAb binding to mutant peptides.
Figure 16 shows the production of anti-IgE antibodies in transgenic animals expressing human IgE.

Justice

The terms used in this specification are interpreted as their usual and typical meanings to those skilled in the art. However, the applicant gives the following specific definitions to the following terms.

An antibody chain “substantially identical” with respect to a polypeptide sequence is interpreted as an antibody chain that exhibits at least 70%, 80%, 90% or 95% sequence homology to the reference polypeptide sequence. With respect to a nucleic acid sequence, the term is interpreted as a nucleotide sequence that exhibits at least 85%, 90%, 95% or 97% sequence homology to the reference nucleic acid sequence.

“Identity” or “homology” is part of sequence homology, after the introduction of a gap as necessary to align the sequence and achieve the maximum homology ratio for the entire sequence, without consideration of any conservative substitutions. It means the ratio of amino acid residues in the candidate sequence that matches the residues of the corresponding sequence to be compared. N- or C-terminal extension and insertion are not interpreted as reducing identity or homology. Methods and computer programs for alignment are well known in the art. Sequence homology can be determined using sequence analysis software.

“Antibody” is used in the broadest sense and specifically encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and multispecific antibodies (eg, bispecific antibodies). Antibodies (Ab) and immunoglobulins (Ig) are glycoproteins that retain the same structural characteristics. Antibodies exhibit binding specificity for a specific target, whereas immunoglobulins include both antibodies and other antibody-like molecules that lack target specificity. Native antibodies and immunoglobulins are generally approximately 150,000 Daltons heterotetrameric glycoproteins composed of two identical light (L) chains and two identical heavy (H) chains. Each heavy chain carries a variable domain (V _H ) and multiple constant domains at one end. Each light chain carries a variable domain at one end (V _L ) and a constant domain at the other end. “High affinity” antibody means an antibody that possesses a binding affinity ^{of at least 10 -10} , preferably 10 ^-12.

As used herein, "anti-human IgE antibody" refers to an antibody that binds to human IgE in a manner that inhibits or substantially decreases the binding of human IgE to a high affinity receptor, FcεRI.

“Variable” with respect to the variable domain of an antibody refers to the fact that certain portions of the variable domain in the sequence of the antibody are significantly changed and are used for the specific binding of each specific antibody to a specific target. However, this variability is not evenly distributed throughout the variable domains of the antibody. It is concentrated in three segments called complementarity determining regions (CDRs) known as hypervariable regions in both the light and heavy chain variable domains. The more conserved portion of the variable domain is called the framework (FR). The variable domains of the intrinsic heavy and light chains each contain four FR regions, almost take the form of a β-sheet, and are linked by three CDRs, which form a loop connecting the β-sheets. , In some cases forming part of the β-sheet structure. In each chain, the CDRs are kept close to each other by the FR regions, and together with the CDRs forming different facts, contribute to the formation of the target binding site of the antibody (Kabat et al.). In the present specification, the numbering of immunoglobulin amino acid residues is not otherwise specified, Kabat et al., Sequences of Proteins of Immunological Interest, National Institute of Health, Bethesda, Ad. It is performed according to the immunoglobulin amino acid residue numbering system of 1987.

“Antibody fragment” refers to a portion of a full-length antibody, usually a target binding or variable region. Examples of antibody fragments are Fab, Fab', F(ab') ₂ , Fv fragments. “Functional fragments or analogs” of antibodies are compounds that possess qualitative biological activity in common with full-length antibodies. For example, a functional fragment of an anti-IgE antibody is a fragment capable of binding IgE immunoglobulin in a manner that prevents or substantially reduces the ability of an IgE immunoglobulin molecule to bind to a high affinity receptor, FcεRI. In the present specification, the "functional fragment" in relation to an antibody refers to an Fv, F(ab), F(ab') ₂ fragment. The “Fv” fragment is the smallest antibody fragment that retains complete target recognition and binding sites. _{This region is composed of a dimer (V H} -V _L dimer) of one heavy chain and one light chain variable domain with a tight, non-covalent bond. In this form, the three CDRs of each variable domain interact to define a target binding site on the surface of _{the V H} -V _{L dimer.} Collectively, the six CDRs donate target binding specificity to the antibody. However, a single variable domain (or half of the Fv containing only three CDRs specific to the target) also retains the ability to recognize and bind to the target, although it has a lower affinity than the entire binding site. “Single-chain Fv” or “sFv” antibody fragments contain the V _H and V _L domains of an antibody, where these domains reside on a single polypeptide chain. In general, the Fv polypeptide further comprises a _{polypeptide linker between the V H} and V _L domains, which linker allows the sFv to form the desired structure for target binding.

The Fab fragment carries the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab' fragments are distinguished from Fab fragments by the addition of several residues at the carboxy terminus of the heavy chain CH1 domain carrying one or more cysteines from the antibody hinge region. F(ab') fragments are produced by cleavage of disulfide bonds at the hinge cysteine _{of the F(ab') 2 pepsin cleavage product.} Additional chemical coupling of antibody fragments is known to those of skill in the art.

As used herein, “monoclonal antibody” refers to an antibody obtained from a population of substantially homologous antibodies, that is, the individual antibodies included in this population are identical except for naturally occurring mutations that exist on a small scale. Monoclonal antibodies are very specific and are directed at a single target site. Furthermore, in contrast to conventional (polyclonal) antibody preparations that typically contain different antibodies against different determinants (epitopes), each monoclonal antibody is directed to a single determinant at the target. In addition to specificity, monoclonal antibodies are advantageous in that they can be synthesized by hybridoma cultures that are not contaminated with other immunoglobulins. “Monoclonal” refers to the properties of an antibody obtained from a substantially homologous population of antibodies and is not to be interpreted as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in the present invention can be isolated from phage antibody libraries using well-known techniques. The parental monoclonal antibody used in the present invention may be prepared by the hybridoma method first described in Kohler and Milstein, Nature 256, 495 (1975), or may be prepared by a recombinant method.

“Humanized” forms of non-human (eg, murine) antibodies include chimeric immunoglobulins, immunoglobulin chains or fragments thereof (eg, Fv, Fab, Fab', F( ab') ₂ or another target-binding subsequence of the antibody). In general, humanized antibodies comprise substantially all of at least one, typically two, variable domains, wherein all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin, and all or almost all of the FR regions It corresponds to the FR region of the human immunoglobulin consensus sequence. Humanized antibodies typically also comprise at least a portion of an immunoglobulin constant region (Fc) of a selected human immunoglobulin template.

“Cell,” “cell line,” and “cell culture” include progeny. Also, due to deliberate or accidental mutations, not all progeny are exactly matched in DNA content. Variant progeny that retain the same function or biological properties, selected from the initially transformed cell, are included. The "host cell" used in the present invention is generally a prokaryotic or eukaryotic host.

The "transformation" of a cell organism with DNA means that the DNA introduced into the organism is capable of replicating either as an extrachromosomal element or by chromosomal integration. “Transfection” of a cell organism with DNA refers to the taking up of DNA, eg, an expression vector, by cells or microorganisms, regardless of whether any coding sequence is actually expressed. The terms "transfected host cell" and "transformed host cell" mean cells into which DNA has been introduced. These cells are called "host cells" and are either protozoan or eukaryotic. Typical prokaryotic host cells are various strains of E. coli. Typical eukaryotic host cells are mammalian cells such as Chinese hamster ovary cells or cells of human origin. The introduced DNA sequence may be derived from the same species as the host cell or from a different species, or may be a hybrid DNA sequence containing some foreign DNA and some homologous DNA.

“Vector” refers to a DNA construct containing a DNA sequence operably linked to an appropriate control sequence capable of inducing the expression of DNA in an appropriate host. Such regulatory sequences include an inducible promoter that performs transcription, a selective operator sequence that controls transcription, a sequence that encodes an appropriate mRNA ribosome binding site, and a sequence that regulates the termination of transcription and translation. Vectors can be plasmids, phage particles, or potential genomic inserts. A vector transformed into a suitable host replicates and functions independently of the host genome, or in some cases integrates into the genome itself. In this specification, “plasmid” and “vector” are frequently used synonymously, because plasmid is the most commonly used form of vector. However, the present invention includes other types of vectors that perform equivalent functions and are known in the art.

“Regulatory sequence” means a DNA sequence required for the expression of a coding sequence operably linked in a particular host organism. Regulatory sequences suitable for eukaryotes include, for example, promoters, optionally operator sequences, ribosome binding sites. Prokaryotic cells are known to use promoters, polyadenylation signals, and enhancers. DNA for a presequence or secretory leader can be operably linked to DNA for a polypeptide when expressed as a preprotein that participates in the secretion of the polypeptide; A promoter or enhancer is operably linked to the coding sequence when it affects the transcription of the sequence; Or the ribosome binding site is operably linked to the coding sequence when it affects the transcription of the sequence; Or the ribosome binding site is operably linked to a coding sequence when arranged to facilitate translation. In general, "operable linkage" means that the DNA sequence to which it is linked is contiguous, contiguous in the case of a secretory leader, and in the reading phase. However, enhancers do not need to be contiguous.

For therapeutic purposes, “mammal” means any animal classified as a mammal, including humans, livestock, non-human primates, zoos, sports or pets, for example dogs, horses, cats, cattle, and the like.

In the context of a polypeptide, “labeled epitope” as used herein refers to a polypeptide fused to an “epitope tag”. The epitope tag polypeptide retains enough residues to provide an epitope short enough that the antibody can be made, but not interfere with the activity of the polypeptide. Suitably, the epitope tag is quite unique such that the antibody does not substantially cross-react with other epitopes. Suitable tag polypeptides have at least 6 amino acid residues, generally approximately 8-50 amino acid residues (preferably approximately 9-30 residues). Examples include influenza HA tag polypeptide and its antibody 12CA5 (Field et al, Mol Cell. Biol. 8: 2159-2165 (1988)); c-myc tag with 8F9, 3C7, 6E10, G4, B7, 9E10 antibodies (Even et al., Mol Cell. Biol. 5(12): 3610-3616(1985)); Herpes Simplex virus glycoprotein D (gD) tag 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), which increases the serum half-life of the IgG molecule in vivo.

As used herein, “label” 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 may be detected by itself (eg, a radioisotope label or a fluorescent label), or, in the case of an enzymatic label, may catalyze chemical modification of a detectable substrate compound or composition.

As used herein, “solid phase” refers to a non-aqueous matrix to which the antibody of the present invention can be attached. Examples of solid phases encompassed by the present invention are solid phases partially or wholly formed of glass (e.g., controlled pore glass), polysaccharides (e.g., agarose), polyacrylamide, polystyrene, polyvinyl alcohol, silicone. In certain embodiments, the solid phase may comprise wells of a test plate; In another embodiment, it is a purification column (eg, affinity chromatography column).

As used herein, “IgE-mediated disease” refers to a disease or abnormality characterized by overproduction and/or hypersensitivity to immunoglobulin IgE. Specifically, it is interpreted to include anaphylactic hypersensitivity reactions and abnormalities associated with atopic allergies, such as asthma, allergic rhinitis & conjunctivitis (hay fever), eczema, urticaria, atopic dermatitis, and food allergies. Severe physiological conditions of anaphylactic shock caused by bee stings, snake bites, food or pharmaceuticals, for example, are also within the scope of the term.

Production of antibodies

Starting or “parent” antibodies can be prepared using techniques known in the art for producing such antibodies. Typical methods of producing starting antibodies are described in more detail in the sections below. These descriptions are possible alternatives for making or preparing parental antibodies and do not limit the methods of producing such molecules.

The binding affinity of the antibody is determined prior to producing the high affinity antibody of the present invention. In addition, the antibody may be subjected to other biological activity tests, for example to evaluate its efficacy as a therapeutic agent. These assays are known in the art and depend on the target and intended use of the antibody.

In order to select an antibody that binds to a specific epitope (eg, an antibody that blocks the binding of IgE to a high affinity receptor), "Antibodies: A Laboratory Manual" (Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988)) Conventional cross-linking tests such as those described in can be performed. Alternatively, epitope mapping can be performed to determine if the antibody binds to the epitope of interest. Optionally, the binding affinity of an antibody (if the homolog is from a different species) to a homolog of the target used to produce the antibody can be assessed using techniques known in the art. In one embodiment, the other species is a non-human mammal to which the antibody is administered in a preclinical study. Thus, these species are non-human primates, such as rhesus, cynomolgus, baboon, chimpanzee, and macaque. In other embodiments, these species are, for example, rodents, cats or dogs. Parental antibodies are modified in accordance with the present invention to produce antibodies that have a higher or stronger binding affinity for the target than the parental antibody. Antibody specificity is due to the unique interface formed between the antibody and its target; The surfaces complement each other to generate a unique fit (Jones, S. & Thornton, J. M. (1996) Proc. Natl. Acad. Sci. USA 93: 13-20). By further enhancing the contact along this interface, the overall affinity can be increased due to the reduction in the energy cost required to promote association of the binding partner.

The binding surface of an antibody is generally composed of six complementarity determining regions (CDRs), which are loops extending from the core. These CDRs are composed of amino acids that have unique sequences for binding to specific targets. In order to increase the affinity of an antibody for an antigen, the environment around these amino acids must be changed more favorably by introducing or enhancing various non-covalent forces, which ultimately change the energy of the interaction. Reduced, resulting in a higher affinity.

Van der Waals forces are non-covalent interactions created between two electrically neutral molecules (Voet, D. & Voet, J. G. (1990) Biochemistry John Wiley and Sons, NY, NY). Bonding can be created between two surfaces from electrostatic interactions resulting from permanent or introduced dipoles. These dipoles may exist along the ends of the α-helix or around polar amino acids. Increasing the total of van der Waals forces along the mating interface leads to a more favorable mating.

The introduction of hydrogen bonds also increases the specificity of the interaction between the antibody and the antigen. Conventional donors and acceptors involved in hydrogen bonding are nitrogen, oxygen, and sulfur atoms, and amino acids are mainly composed of these atoms (See Voet, et al., supra). Since hydrogen bonds tend to crosslink only short distances (typically 2.7 to 3.1 Å), the binding partners must be close so that these interactions can occur. Thus, one way to improve affinity is to establish hydrogen bonds by bringing the potential donor and acceptor molecules into close contact.

Finally, the enhancement of the hydrophobic interaction also increases the favorable energy between the two binding partners. Non-polar moieties located proximate to the binding surface are surrounded by other non-polar moieties, and thus exist in a friendly environment. By satisfying the burial of the non-polar side chain, the energy of the interaction is advantageous for the strong bonding interface.

Interactions that stabilize the protein-protein interface reduce the energy cost of maintaining these contacts, thereby increasing overall affinity. By improving the environment around individual amino acids located near the binding interface, a more favorable environment is induced resulting in a higher binding affinity. For this reason, by introducing friendly contact and improving the interface through additional supplementation, the overall binding interaction between the antibody and the antigen is significantly improved.

Suitably, the resulting high affinity antibody retains a binding affinity that is at least 10 times, or at least 20 times, or at least 500 times, or 1000 to 5000 times higher than that of the parent antibody for the target. The degree of improvement in the required or required binding affinity depends on the initial binding affinity of the parent antibody.

In general, the method of making a high affinity antibody from a parental antibody involves the following steps:

1. A step of obtaining or selecting a parental antibody that binds to a target target and contains a heavy and light chain variable domains. This step is performed by traditional hybridoma techniques, phage-display techniques, or any other method of producing target specific antibodies.

2. Selecting a framework sequence close to the parent framework, preferably a human template sequence, from the sequence. The template is selected based on the comparative overall length, the size of the CDRs, the amino acid residues located at the junction between the framework and the CDRs, and overall homology. The selected template may be a mixture of one or more sequences, or may be a consensus template.

3. Generating a clonal library by performing random amino acid substitutions at all possible CDR positions. For example, it is also possible to generate a library of framework substitutions by randomly replacing amino acids with all possible amino acids in a human framework template that is adjacent to the CDR or affects binding or folding. These backbone substitutions can assess their potential effects on target binding and antibody folding. Substitution of amino acids in the framework may be performed simultaneously or sequentially with substitution of amino acids in CDRs. One way to generate a library of variants by synthesizing oligonucleotides.

4. Formula: FRH1-CDRH1-FRH2-CDRH2-FRH3-CDRH3-FRH4(1) and FRL1-CDRL1-FRL2-CDRL2-FRL3-CDRL3-FRL4(II), and the heavy chain produced in step (3) and/ Or constructing an expression vector comprising a light chain variant, wherein FRL1, FRL2, FRL3, FRL4, FRH1, FRH2, FRH3, FRY represent variants of the framework template light and heavy chain sequences selected in step 3, and CDRs are of parental antibody CDRs. The variant CDR is shown. An example of a vector carrying such light and heavy chain sequences is shown in FIG. 1.

5. Screening for enhanced binding affinity in a clone library for a specific target and clones that bind to the target. Clones that bind with higher affinity than the parent molecule can be screened. Optimal high affinity candidates retain the greatest possible binding affinity compared to the parental antibody, preferably at least 20 times, 100 times, 1000 times, or 5000 times or more. If the selected variant has certain undesired amino acids, such as introduced glycosylation sites or potentially immunogenic sites, these amino acids are replaced with more favorable amino acid residues and binding affinity is reassessed.

In addition, using such a method, it is also possible to generate a high affinity antibody from a fully human parental antibody by randomly replacing only the CDR regions while leaving the human skeleton as it is.

Due to the improved high-efficiency screening technology and vector as shown in FIG. 1, those skilled in the art can quickly and efficiently screen a comprehensive substitution library at all sites of a given CDR and/or framework region. By simultaneously randomly substituting all amino acids at all positions, it is possible to screen for possible combinations with markedly increased affinity due to synergistic effects not expected or confirmed by individual substitutions.

Parental antibody preparation

Target preparation

Soluble targets or fragments thereof can be used as immunogens to produce antibodies. Antibodies are directed at the target of interest. Suitably, the target is a biologically important polypeptide, and administration of the antibody to a mammal suffering from a disease or condition can result in therapeutic benefits in the mammal. However, antibodies can also 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. One target of the present invention is IgE. Whole cells can be used as immunogens to produce antibodies. Targets can be produced recombinantly or can be prepared using synthetic methods. Targets can also be separated from natural sources.

Examples of antigens used to produce the antibodies of the invention are the polypeptides and polypeptide fragments of the invention, including epitopes A and/or B. Polypeptides used to immunize animals can be obtained by standard recombinant, chemical synthesis, or purification methods. As is known in the art, to increase immunogenicity, antigens can be conjugated to a carrier protein. Carriers commonly used include keyhole limpet hemocyanin (KLH), serum thyroid globulin, bovine serum albumin (BSA), and tetanus toxoid. Not limited. Thereafter, an animal (eg, mouse, rat, or rabbit) is immunized using the bound peptide. In addition to these carriers, well-known adjuvants can be administered together with an antigen to promote the induction of a strong immune response.

Polyclonal antibody

Polyclonal antibodies are generally produced in non-human mammals by subcutaneous (se) or intraperitoneal (ip) injection of an associated target in combination with an adjuvant. A number of pathogens capable of inducing an immune response are well known in the art.

Animals are immunized against targets, immunogenic conjugates or derivatives by conjugating proteins or conjugates (for rabbits or mice, respectively) with Freund's complete adjuvant and injecting the solution intradermally. After 1 month, these animals promote efficacy by subcutaneous injection at multiple sites of 1/5 to 1/10 of the original amount of peptide or conjugate contained in Freund's incomplete adjuvant. After 7-14 days, these animals are bled and the serum is tested for antibody titer. Animals promote potency until the titer plateau.

Selected mammalian antibodies normally retain a sufficiently strong binding affinity for the target. For example, the antibody binds to human anti-IgE with a binding affinity (Kd) value ^{of approximately 1 x 10 -8 M.} Antibody affinity includes saturation binding; Enzyme-linked immunoabsorbant assay (ELISA); Competitive tests (e.g. radioimmunoassays) can be determined.

In order to screen for antibodies that bind to the target target, a conventional cross-linking assay as described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988) is performed. I can. Alternatively, for example Champe, et al. J. Biol. Chem. 270: Epitope genetic mapping can be performed as described in 1388-1394 (1995) to determine binding.

Monoclonal antibody

Monoclonal antibodies are antibodies that recognize a single antigenic site. Because of their uniform specificity, monoclonal antibodies are generally more useful than polyclonal antibodies that recognize a large number of different antigenic sites. Monoclonal antibodies may be prepared by the hybridoma method first described in Kohler and Milstein, Nature 256, 495 (1975), or may be prepared by a recombinant method.

In the hybridoma method, mice or other suitable host animals, such as rodents, are immunized as described above to induce lymphocytes that produce or are capable of producing antibodies that specifically bind to the protein used for immunization. Alternatively, lymphocytes may be immunized in vitro. Thereafter, the lymphocytes are fused with myeloma cells using an appropriate fusing agent, for example, polyethylene glycol to form hybridoma cells (Goding, Monoclonal Antibodies: Principals and Practice, pp. 590-103 (Academic). Press, 1986)).

The hybridoma cells thus prepared are inoculated and grown in an appropriate culture medium containing one or more substances that inhibit the growth or survival of unfused parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for hybridomas is typically hypoxanthine, aminopterin, thymidine ( thymidine) (HAT medium), a substance that prevents the growth of HGPRT-deficient cells. Preferred myeloma cells efficiently fuse the selected antibody-producing cells, support stable and high-level antibody production by these cells, and are sensitive to media such as HAT media. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been reported (Kozbar, J.: Immunol. 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).

Once hybridoma cells producing antibodies of the desired specificity, affinity and/or activity are identified, these clones can be subcloned and grown by standard methods by limiting the dilution procedure (Goding, Monoclonal Antibodies:: Principals and Practice). , pp. 59-103, Academic Press, 1986)). A culture medium suitable for this purpose is included. Monoclonal antibodies secreted by these subclones are appropriately separated from the culture medium by conventional immunoglobulin purification procedures, such as protein A sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography. .

DNA encoding these monoclonal antibodies is conveniently separated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of these monoclonal antibodies). do. Hybridoma cells function as a source of this DNA. Once isolated, the DNA is placed in an expression vector, and the expression vector is transferred to a host cell, for example, E. coli cells, NS0 cells, Chinese hamster ovary (CHO) cells, or myeloma cells, and the recombinant host cells To achieve the synthesis of monoclonal antibodies. In addition, DNA is replaced with, for example, a coding sequence for human heavy and light chain constant domains instead of homologous murine sequences (US Pat. No. 4,816,567; Morrison et al., Proc. Natl Acad. Sci. USA 81: 6851 ( 1984)), or by covalent linking to an immunoglobulin polypeptide.

Humanized antibody

Humanization is a technique for producing chimeric antibodies in which a substantially circular human variable domain has been replaced with a corresponding sequence derived from a non-human species. Humanized antibodies are introduced into one or more amino acid residues from non-human sources. These non-human amino acid residues are referred to as “import” residues, which are typically derived from “import” variable domains. Humanization is essentially by substituting the corresponding sequence in a human antibody with a non-human CDR or CDR sequence, such as Winter and his colleagues (Jones et al, Nature 321: 522-525 (1986); Riechman et al., Nature 332: 323). -327 (1988); Verhoeyens et al., Science 239: 1534-1536 (1988)). In the present invention, in the humanized antibody, some CDR residues and some FR residues may be substituted with residues derived from similar sites in the murine antibody.

The selection of the human variable domains (both light and heavy chains) used to make humanized antibodies is very important in reducing antigenicity. According to the so-called “best fit” method, the sequence of the variable domains of a non-human antibody is compared with a library of known human variable domain sequences. Thereafter, the human sequence closest to the sequence of the non-human parental antibody is accepted as a 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 uses a specific backbone derived from the consensus sequence of all human antibodies of a specific subgroup of the light or heavy chain. The same backbone may be used for several different humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89: 4285 (1992); Presta et al., J. Immunol. 151: 2623 (1993)) ).

Antibody fragment

Various techniques have been developed for the production of antibody fragments. Typically, these fragments are derived through proteolytic cleavage of the original antibody (Morimoto et al., Journal of Biochemical and Biophysical Methods 24: 107-117 (1992); Brennan et al., Science 229: 81 (1985) )). However, these fragments can also be produced directly by recombinant host cells. For example, antibody fragments can be isolated from antibody phage libraries. Alternatively, the F(ab') ₂ -SH fragment can be recovered directly from E. coli and chemically bound to form the F(ab') ₂ fragment (Carter et al., Bio/Technology 10: 163-167 (1992)). Alternatively, F(ab') ₂ fragments can be isolated directly from recombinant host cell culture. Other techniques for the production of antibody fragments are apparent to those of skill in the art. In another embodiment, the antibody of choice is a single chain Fv fragment (scFv) (PCT patent application WO 93/16185).

Preparation of high affinity antibody

Once the parental antibody has been identified and isolated, one or more amino acid residues can be altered in one or more variable regions of the parental antibody. Alternatively, or additionally, one or more substitutions of framework moieties may be introduced into the parental antibody, where they enhance the binding affinity for the antibody, for example human IgE. Examples of changing framework region residues include residues that non-covalently bind directly to the target (Amit et al. Science 233: 747-753 (1986)); Residues that interact with the CDR or affect the structure of the CDR (Chothia et al. J. Mol. Biol. 196: 901-917 (1987)); And/or _{a residue that participates in the V L} -V _H interface (EP 239 400 B1). In certain embodiments, modification of one or more of these framework region residues results in an enhancement of the binding affinity of the antibody to the target of interest.

Changes in the biological properties of the antibody include (a) the structure of the polypeptide backbone in the substitutional region, eg in the form of a sheet or helix; (b) the charge or hydrophobicity of the molecule at the target site; Or (c) by selecting a substitution that differs significantly in its effect on maintaining the size of the side chain. Non-conservative substitutions involve the process of replacing one member of these classes with a member of another class.

Nucleic acid molecules encoding amino acid sequence variants are prepared by a variety of methods known in the art. These methods include, but are not limited to, oligonucleotide-mediated (or site specific) mutagenesis, PCR mutagenesis, cassette mutagenesis of previously prepared variants or non-variant variants of species-dependent antibodies. A suitable method of producing variants is oligonucleotide-mediated synthesis. In certain embodiments, the antibody variant has only a single hypervariable region residue substituted, e.g., has approximately 2 to 15 hypervariable region substitutions.

One way to generate a variant library is oligonucleotide mediated synthesis according to the scheme shown in FIG. 2. Three oligonucleotides consisting of approximately 100 nucleotides each can be synthesized over the entire light chain or heavy chain variable region. Each oligonucleotide is (1) _{a 60 amino acid stretch produced by triplet (NNK) 20} , where N is any nucleotide and K is G or T; (2) the following oligos, or may contain approximately 15-30 nucleotide overlaps with the vector sequence at each end. After annealing of these three oligonucleotides in the PCR reaction, the polymerase fills the opposite strands to produce a complete double-stranded heavy or light chain variable region sequence. Amino acids can be substituted only in certain CDRs or framework regions by adjusting the total of triplet to an arbitrary repeat length and selecting their positions within the oligonucleotide. By using (NNK), a total of 20 amino acids are possible at each position of the encoded variant. The overlapping sequence of 5-10 amino acids (15-30 nucleotides) is not substituted, but it may be selected to be included within the stacking region of the backbone, or may be substituted in separate or subsequent synthetic processes. Methods for synthesizing oligonucleotides are well known in the art and are also commercially available. Methods of producing antibody variants from these oligonucleotides, such as PCR, are also well known in the art.

The heavy and light chain variant libraries that are differentiated at random positions in the sequence can be constructed in any expression vector, for example, a bacteriophage, especially the vector of Figure 1, each of which has DNA encoding a specific heavy and light chain variant. .

Following production of antibody variants, the biological activity of these variants is determined by comparison with the parent antibody. As mentioned above, this entails the process of determining the binding affinity of the variant for the target. There are a number of highly efficient methods for rapidly screening antibody variants for their ability to bind to the target of interest.

One or more antibodies selected from this initial screening can then be screened for improved binding affinity compared to the parent antibody. One common method for determining binding affinity is to use a BIAcore ^TM surface plasmon resonance device (BlAcore, Inc.) to evaluate the association and dissociation rate constants. will be. Activate the biosensor chip for covalent binding of the target according to the manufacturer's (BlAcore) instructions. Thereafter, the target is diluted and injected into the chip to obtain a signal (reaction unit (RU)) of the immobilized material. Since the signal (RU) is proportional to the volume of the immobilized material, it represents the range of immobilized target densities in the matrix. Separate data are fitted to a one-site model to obtain k _off +/- sd (standard deviation of dimensions). Calculate the pseudo-first order rate constant (K _S ) for each binding curve, and determine the position in the coordinates as a function of the protein concentration, and k _on +/se (the standard of fit). Error). Equilibrium dissociation constants for binding K _D' S is k _off /k _on and is calculated from the SPR dimension. Since the equilibrium dissociation constant K _D is _{inversely proportional to k off} , the affinity enhancement can be assessed under the assumption that the _{binding ratio (k on) is constant in all variants.}

Candidates with high affinity can optionally perform one or more additional biological activity assays to ascertain whether antibody variants with enhanced binding affinity retain the desired therapeutic properties. For example, in the case of anti-IgE antibodies, those that block the binding of IgE to the receptor and inhibit the release of histamine can be screened. Optimal antibody variants retain the ability to bind to the target with much higher binding affinity than the parent antibody.

The antibody variants thus selected may be further modified depending on the intended use of the antibody. Such modifications may involve further modifications of the amino acid sequence, fusion to heterogeneous polypeptides, and/or covalent modifications as detailed below. For example, cysteine residues that are not involved in maintaining the proper structure of antibody variants are generally substituted with serine to improve the oxidative stability of the molecule and prevent abnormal crosslinking. Conversely, cysteine bonds can also be added to the antibody to improve its stability (especially when the antibody is an antibody fragment such as an Fv fragment).

vector

In addition, the present invention provides an isolated nucleic acid encoding the antibody variant described herein, a vector and host cell comprising the nucleic acid, and a recombinant technology for the production of the antibody variant. For recombinant production of antibody variants, nucleic acids encoding them are isolated and inserted into cloning vectors for further cloning (amplification of DNA) or expression. DNA encoding the antibody variant is conveniently isolated and sequenced using conventional procedures (eg, using an oligonucleotide probe capable of specifically binding to genes encoding the heavy and light chains of the antibody variant).

A number of vectors are available. Vector elements generally include, but are not limited to, signal sequences, origins of replication, one or more marker genes, enhancer elements, promoters, transcription termination sequences.

Phage expression vector shown in Figure 1 is a commonly used M13 vector; It is composed of M13's own gene III viral secretion signal for rapid secretion and screening of appropriate binding specificity and minimal affinity criteria in variant Fabs. Since the vector does not use the entire gene III sequence, it is not displayed on the surface of the bacterial cell, but rather the Fab is secreted into the periplasmic space. Alternatively, these Fabs can be expressed and isolated in the cytoplasm. The heavy and light chains each have their own viral secretion signals, but are expressed dependently from one strong inducible promoter.

The vector shown in FIG. 1 provides a His tag and a myc tag for convenient purification as well as detection. As those skilled in the art will appreciate, these Fabs may be expressed independently from separate promoters, or the secretion signal need not be a selected viral sequence and may be a prokaryotic or eukaryotic signal sequence suitable for secretion of antibody fragments from the selected host cell. In addition, the heavy and light chains may exist in different vectors.

A. Signal sequence component

Antibody variants of the present invention can be produced in a recombinant manner. Variants may also be expressed as a fusion polypeptide fused with a heterogeneous polypeptide, preferably a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide. Suitably, the selected heterogeneous signal sequence is recognized and processed by the host cell (ie, cleaved by the signal peptidase). For prokaryotic host cells that do not recognize and process the native antibody signal sequence, the signal sequence may be, for example, alkaline phosphatase, penicillinase, Ipp, or a heat-stable enterotoxin II leader ( heat-stable enterotoxin II leader). Alternatively, in the case of the vector of Figure 1, the selected signal sequence was a viral signal sequence from gene III. For yeast secretion, intrinsic signal sequences include, for example, yeast invertase leader, α-factor leader ( Saccharomyces ) and Kluyveromyces α-factor leader. ), an acid phosphatase leader, a C. albicans glucoamylase leader, or, for example, a signal described in WO 90/13646. In mammalian cell expression, mammalian signal sequences and viral secretion leaders such as herpes simplex gD signals are available. The DNA for these precursor regions is ligated in the reading frame to the DNA encoding the antibody variant.

B. Origin of the replication component

In general, a vector carries a nucleic acid sequence that allows the vector to replicate in one or more selected host cells. In general, the sequence allows the vector to replicate independently of the host chromosomal DNA, and has an origin of replication or an automatic replication sequence. Such sequences are well known in a variety of bacteria, yeasts, and viruses. The origin of replication from plasmid pBR322 is suitable for most Gram-negative bacteria, the 2 μ plasmid origin is suitable for yeast, and various viral origins (SV4O, polyoma, adenovirus, VSV or BPV) are vectors in mammalian cells. Suitable for In general, the origin of the replication component is not required for mammalian expression vectors (the SV40 origin can typically be used only if the replication component carries an early promoter).

C. Selective Gene Components

The vector carries a selection gene called a selectable marker. Typical selection genes include (a) proteins that donate resistance to antibiotics or other toxins, such as ampicillin, neomycin, methotrexate, or tetracycline; (b) proteins that compensate for auxotrophic deficiencies; Or (c) encoding a protein that provides important nutrients lacking in the complex medium (eg, a gene encoding D-alanine racemase for Bacillus).

One example of a screening scheme uses drugs that stop the growth of host cells. Cells successfully transformed with heterogeneous genes produce proteins that donate drug resistance and thus overcome this screening task. Examples of such dominant screening use the drugs neomycin, mycophenolic acid, and hygromycin.

Other examples of selectable markers suitable for mammalian cells include markers that allow identification of cells capable of inhaling antibody nucleic acids, such as DHFR, thymidine kinase, metallothionein- I and II, preferably a primate metallothionein gene, adenosine deaminase, ornithine decarboxylase, and the like.

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 wild-type DHFR is used is a Chinese hamster ovary (CHO) cell line that lacks DHFR activity.

Alternatively, host cells transformed or co-transformed with a DNA sequence encoding an antibody, wild-type DHFR protein, other selectable marker, e.g., aminoglycoside 3'-phosphotransferase (APH) (particularly , Wild-type host carrying endogenous DHFR) is an aminoglycosidic antibiotic, e.g., a selection agent for a selectable marker such as kanamycin, neomycin, or G418. (US Pat. No. 4,965,199) (selection agent) can be selected by cell growth in a medium containing.

A selection gene suitable for use in yeast is the trp1 gene present in the yeast plasmid Yrp7 (Stinchcomb et al., Nature 282: 39 (1979)). The trp1 gene is a yeast mutant strain lacking the ability to grow on tryptophan, for example ATCC No. Selection markers for 44076 or PEP4-1 are provided (Jones, Genetics 85: 12 (1977)). The presence of a trp1 disorder in the yeast host cell genome provides an effective environment for detecting transformation by growth in the absence of tryptophan. Similarly, Leu2-deficient yeast strains (ATCC 20, 622 or 38, 626) are supplemented by known plasmids carrying the Leu2 gene.

D. Promoter Components

Expression and cloning vectors generally carry a promoter that is recognized by the host organism and is operably linked to the antibody nucleic acid. Promoters suitable for use in prokaryotic hosts are the phoA promoter, β-lactamase and lactose promoter systems, alkaline phosphatase, tryptophan (trp) promoter systems, hybrid promoters (eg, tac promoter) and the like. However, other known bacterial promoters are also suitable. Promoters used in bacterial lineages may also have Shine-Dalgarno (S.D.) sequences operably linked to DNA encoding the antibody.

Promoter sequences for eukaryotes are known. Almost all eukaryotic genes have an AT-rich region located approximately 25 to 30 bases upstream from the site where transcription is initiated. Another sequence found 70 to 80 bases upstream from the initiation site of transcription of many genes is the CNCAAT region, where N is any nucleotide. The AATAAA sequence is present at the 3'end of most eukaryotic genes, which is a signal for the addition of a poly A tail at the 3'end of the coding sequence. All of these sequences are appropriately inserted into eukaryotic expression vectors.

Examples of suitable promoter sequences for use in yeast hosts include 3-phosphoglycerate kinase or other glycolytic enzymes such as enolase, glyceraldehyde-3-phosphate. Dehydrogenase (glyceraldehyde3-phosphate dehydrogenase), hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase (glucose-6- phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, glucokinase ( glucokinase).

Other yeast promoters are alcohol dehydrogenase 2, isocytochrome C, and acid phosphatase. , A degrading enzyme associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for the use of maltose and galactose. Vectors and promoters suitable for use in yeast expression are further detailed in EP 73,657. Suitably, yeast enhancers are also used in conjunction with yeast promoters.

Antibody transcription from vectors in mammalian host cells is, for example, polyoma virus, fowlpox virus, adenovirus (e.g., adenovirus 2), bovine papilloma virus, avian sarcoma virus. (avian sarcoma virus), cytomegalo virus (cytomegalo virus), retrovirus (retrovirus), hepatitis B virus (hepatitis-B virus), most preferably simian virus 40 (Simian virus 40) (SV40) A promoter obtained from the genome; Heterogeneous mammalian promoters, such as actin promoters or immunoglobulin promoters; Or regulated by heat-shock promoters, where these promoters must be suitable for the host cell system.

The early and late promoters of the SV40 virus are conveniently obtained as SV40 restriction fragments bearing the SV40 virus origin of replication. The immediate early promoter of human cytomegalovirus is conveniently obtained as a HindIII E restriction fragment. The system for expressing DNA in mammalian hosts using bovine papilloma virus as a vector is U.S. Pat. No. It is described in 4,419,446. A variation of the system is U.S. Pat. No. It is described in 4,601,978. Alternatively, human β-interferon cDNA was expressed in mouse cells under the control of the thymidine kinase promoter obtained from herpes simplex virus. Alternatively, a rous sarcoma virus long terminal repeat can be used as a promoter.

E. Enhancer Element Components

The transcription of DNA encoding the antibodies of the invention by higher eukaryotes is frequently increased by inserting an enhancer sequence into the vector. A number of enhancer sequences are known from mammalian genes (globin, elastase, albumin, α-fetoprotein, insulin). However, typically, enhancers are used from eukaryotic cell viruses. Examples are SV40 enhancer (bp 100-270), cytomegalovirus early promoter enhancer at the posterior of the origin of replication, polyoma enhancer at the rear of the replication origin, and adenovirus enhancer. See Yaniv, Nature 297: 17-18 (1982) for enhancer elements for activation of eukaryotic promoters. The enhancer can be truncated to the vector at the 5'or 3'position of the antibody-encoding sequence, but is preferably located 5'from the promoter.

F. Transcription Termination Components

Expression vectors used in eukaryotic host cells (cells with nuclei from yeast, fungi, insects, plants, animals, humans, or other multicellular organisms) may contain sequences necessary for transcription termination and mRNA stabilization. These sequences are typically obtained from the 5'end, sometimes from the 3'untranslated region of eukaryotic or viral DNA or cDNA. These regions carry nucleotide segments that are transcribed as polyadenylation fragments in the untranslated region of the mRNA encoding the antibody. One useful transcription termination component is the bovine growth hormone polyadenylation region (WO94/11026).

Selection and transformation of host cells

Suitable host cells for cloning or expressing DNA in the vectors of the invention are prokaryotic cells, yeast, or higher eukaryotic cells. Suitable prokaryotes for this purpose is Bacillus (Bacillus), Pseudomonas bacterium (Pseudomonas), streptomycin MRS (Streptomyces), as well as Gram-negative and Gram-positive organisms, for, example, E. coli (E. coli), Enterobacter (Enterobacter) a beanie El ah Species (Erwinia), keulrepeu when Ella bacteria (Klebsiella), Proteus (Proteus), Salmonella (Salmonella), Serratia genus (Serratia), Shigella intestinal bacteria (enterobacteria), such as (Shigella). One preferred E. coli cloning host is E. coli 294 (ATCC 31,446), but other strains such as E. coli B, E. coli X1776 (ATCC 31,537) ), E. coli ( E. coli ) W3110 (ATCC 27,325) is also suitable. These are non-limiting examples.

In addition to prokaryotes, eukaryotic microorganisms such as fibrotic fungi or yeast are suitable as cloning or expression hosts for antibody-encoding vectors. Saccharomyces cerevisiae is the most commonly used among lower eukaryotic host microorganisms. However, a number of other genera, species, strains, such as Schizosaccharomyces pombe ; Kluyveromyces ; Candida (Candida); Trichoderma ; Neurospora crassa ; Fibrous fungi, for example, Neurospora , Penicillium , Tolypocladium , Aspergillus host (eg, Aspergillus nidulans) ) And Aspergillus niger ) are conveniently available and used in the present invention.

Host cells suitable for expression of glycosylated antibodies are derived from multicellular organisms. In principle, any higher eukaryotic cell culture can be used, whether derived from vertebrate or invertebrate cultures. Examples of invertebrate cells are plant and insect cells (Luckow et al., Bio/Technology 6, 47-55 (1988); Miller et al., Genetic Engineering, Setlow et al. ads. Vol. 8, pp. 277 -279 (Plenam publishing 1986); Mseda et al., Nature 315, 592-594 (1985)). Correspondence from multiple baculovirus strains and variants and hosts such as Spodoptera frugiperda (larvae), forest mosquitoes (Aedes) (mosquitoes), Drosophila melanogaster (clownfish) and silkworm moths ( Bombyx mori ). Replicable insect host cells have been identified. Various viral strains for transfection, such as the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, are publicly available, these viruses According to the invention, it can be used as a virus , in particular for transfection of Spodoptera frugiperda cells. In addition, plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be used as hosts.

Proliferation by culture of vertebrate cells (tissue culture) has become routine (see: Tissue Culture, Academic Press, Kruse and Patterson, eds. (1973)). Examples of useful mammalian host cell lines include monkey kidney; Human fetal kidney cell line; Baby hamster kidney cells; Chinese hamster ovary cells/-DHFR (CHO, Urlaub et ai., Proc. Natl. Acad. Sci. USA 77: 4216 (1980)); Mouse sertoli cells; Human cervical carcinoma cells (HELA); Canine kidney cells; Human lung cells; Human liver cells; Mouse breast tumor; It is an NS0 cell.

Host cells are transformed with the above-described vector for antibody production and cultured in a conventional nutrient medium modified to be suitable for inducing a promoter, selecting a transformant, or amplifying a gene encoding a desired sequence.

The host cells used to produce the antibody variants of the present invention can be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium (MEM, Sigma), RPMI-1640 (Sigma), Dulbecco's Modified Eagle's Medium (DMEM, Sigma) are suitable for culturing host cells. In addition, Ham et al., Meth. Enzymol. 58. 44 (1979); Barnes et al., Anal. Biochem. 102: 255 (1980); U.S. Pat. No. 4,767,704; 4,657,866; 4,560,655; 5,122,469; 5,712,163; Any of the media described in 6,048,728 can be used as a culture medium for host cells. These media may be, as needed, hormones and/or other growth factors (eg insulin, transferrin or epidermal growth factor), salts (eg, X-chloride, where X is sodium, calcium, magnesium); Phosphate), buffers (e.g. HEPES), nucleotides (e.g., adenosine and thymidine), antibiotics (e.g. GENTAMYCIN.TM. drugs), trace elements (typically, inorganic compounds present in final concentrations in the micromolar range). It can be supplemented with glucose or an equivalent energy source. Any other necessary supplements known to those skilled in the art may also be included in suitable concentrations. Culture conditions, for example, temperature, pH, and the like are the same as those previously used in host cells selected for expression, and are apparent to those skilled in the art.

Antibody purification

When using recombinant techniques, antibody variants can be produced intracellularly or in the periplasm, or secreted directly into the medium. When antibody variants are produced intracellularly, as a first step, particulate debris of host cells or lysed fragments is removed, for example by centrifugation or ultrafiltration. Carter et al., Bio/Technology 10: 163-167 (1992) describes a procedure for isolating antibodies secreted into the periplasm of E. coli. Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) for 30 minutes. Cell debris can be removed by centrifugation. When antibody variants are secreted into the medium, the supernatant from these expression systems is generally first concentrated using commercially available protein concentration filters, such as Amicon or Millipore Pellicon ultrafiltration units. Protease inhibitors 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 accidental contaminants.

Antibody compositions prepared from these cells can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being preferred as a purification technique. The suitability of Protein A as an affinity ligand depends on the type and isotype of any immunoglobulin Fc domain present in the antibody variant. Protein A can be used to purify antibodies based on human IgG1, IgG2 or IgG4 heavy chains (Lindmark et al., J. Immunol Meth. 62: 1 13 (1983)). Protein G is recommended for all mouse isotypes and human IgG3 (Guss et al., EMBO J. 5: 1567-1575 (1986)). The matrix to which the affinity ligand is attached is most often agarose, but other matrices are possible. Mechanically stable matrices, such as controlled pore glass or poly(styrenedivinyl)benzene, allow for faster flow rates and shorter processing times than in agarose. When the antibody variant comprises a CH3 domain, Bakerbond ABXTM resin (JT Baker, Phillipsburg, NJ) is useful for purification. , For another technique, for example, for protein purification, ion-classification in the exchange column (ion-exchange column) (fractionation ), precipitated with ethanol (ethanol precipitation), chromatography (anion) in the chromatography, heparin SEPHAROSE ^TM in reverse phase HPLC, a silica Alternatively, chromatography, chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation in a cation exchange resin (e.g., a polyaspartic acid column) may also be used depending on the antibody variants recovered.

After any preliminary purification step, the mixture containing the desired antibody variant and contaminant is preferably subjected to low pH hydrophobic interaction chromatography using an elution buffer of approximately 2.5-4.5 pH at a salt concentration as low as possible (e.g., approximately 0-0.25 M salt). Photography can be performed.

Pharmaceutical composition

Therapeutic compositions of polypeptides or antibodies contain a polypeptide that retains the desired level of purity, such as an optional “pharmaceutically acceptable” carrier, excipient or stabilizer (all referred to as “excipients”) typically employed in the art. , Buffers, stabilizers, preservatives, isotonic agents, non-ionic surfactants, antioxidants, and other additives, can be prepared as freeze-dried compositions or aqueous solutions for storage (Remington's Pharmaceutical Sciences, 16th edition). , A. Osol, Ed. (1980)). These additives should be non-toxic to the recipient at the dosage and concentration used.

Buffers help keep the pH in a range that approximates physiological conditions. Suitably, they are present at concentrations in the range of approximately 2 mM to 50 mM. Buffers suitable for use in the present invention include organic and inorganic acids and salts thereof, such as citrate buffers (e.g., sodium citrate-disodium citrate mixture, citric acid-trisodium citrate mixture, citric acid-sodium citrate mixture, etc.), succinate. Buffer (e.g., succinic acid-sodium succinate mixture, succinic acid-sodium hydroxide mixture, succinic acid-disodium succinate mixture, etc.), tartrate buffer (e.g., tartaric acid-sodium tartrate mixture, tartaric acid-potassium tartrate mixture, tartaric acid-sodium hydroxide mixture, etc.) , Fumarate buffer (e.g., fumaric acid-sodium fumarate mixture, etc.), fumarate buffer (e.g., fumaric acid-sodium fumarate mixture, fumaric acid-disodium fumarate mixture, sodium fumarate-disodium fumarate mixture, etc.), gluconate buffer (e.g. , Gluconic acid-sodium gluconate mixture, gluconic acid-sodium hydroxide mixture, gluconic acid-potassium gluconate mixture, etc.), oxalate buffer (e.g., oxalic acid-sodium oxalate mixture, oxalic acid-sodium hydroxide mixture, oxalic acid-potassium oxalate mixture, etc.) ), lactate buffer (e.g., lactic acid-sodium lactate mixture, lactic acid-sodium hydroxide mixture, lactic acid-potassium lactate mixture, etc.), acetate buffer (e.g., acetic acid-sodium acetate mixture, acetic acid-sodium hydroxide mixture, etc.), and the like. Additionally, phosphate buffers, histidine buffers, trimethylamine salts (eg Tris) are also possible as buffers.

Preservatives can be added in amounts ranging from 0.2%-1% (w/v) to retard microbial growth. Preservatives suitable for use in the present invention include phenol, benzyl alcohol, meta-cresol, methyl paraben, propyl paraben, octadecyldimethylbenzyl ammonium chloride, benzalconium halide (e.g., chloride, bromine Cargo, iodide), hexamethonium chloride, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, and 3-pentanol.

An isotonic agent, also referred to as a "stabilizer", is added to ensure the isotonicity of the liquid composition of the present invention, which includes a polyhydric sugar alcohol, preferably a trihydric or higher sugar alcohol, for example. For example, glycerin, erythritol, arabitol, xylitol, sorbitol, mannitol, and the like are included.

Stabilizers refer to a wide range of excipients in terms of function, from bulking agents to additives that help dissolve the therapeutic agent or prevent denaturation or adhesion to the container wall. Typical stabilizers include polyhydric sugar alcohols (listed above); Amino acids such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, threonine, and the like; Organic sugars or sugar alcohols, including cyclitol, such as inositol, such as lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, myoini Myoinisitol, galactitol, glycerol, and the like; Polyethylene glycol; Amino acid polymer; Sulfur-bearing reducing agents such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, α-monothioglycerol, sodium thio Sodium thio sulfate; Low molecular weight polypeptides (ie, <10 residues); Proteins such as human serum albumin, bovine serum albumin, gelatin or immunoglobulins; Hydrophilic polymers such as polyvinylpyrrolidone monosaccharide (e.g. xylose, mannose, fructose, glucose; disaccharides such as lactose) , Maltose, sucrose; trisaccacharide, e.g. raffinose; polysaccharide, e.g. dextran; stabilizer is 0.1 to 10,000 weight ratio of active protein weight Exists in scope.

Non-ionic surfactants or detergents (“wetting agents”) are added to help stabilize the therapeutic as well as protect the therapeutic protein from agitation-induced aggregation, which is the shear plane in which the composition is pressed without denaturing the protein. (shear surface). Suitable non-ionic surfactants include polysorbate (20, 80, etc.), polyoxamer (184, 188 etc.), Pluronic polyol, polyoxyethylene sorbitan monoether ( TWEEN, TWEEN, etc.). The non-ionic surfactant is present in the range of approximately 0.05 mg/mL to 1.0 mg/mL, preferably approximately 0.07 mg/mL to 0.2 mg/mL.

Additional other excipients include bulking agents (eg starch), chelating agents (eg EDTA), antioxidants (eg ascorbic acid, methionine, vitamin E), co-solvents, and the like. The compositions of the present invention may further contain one or more active compounds as necessary for the particular indication being treated, preferably active compounds having complementary activities that do not adversely affect each other. For example, it may be desirable to provide additional immunosuppressants. Suitably, these molecules are present in an effective amount combination that meets the intended purpose. These active ingredients are, for example, microcapsules prepared by coacervation technology or interfacial polymerization, for example colloidal drug delivery systems (e.g. liposomes, albumin microspheres, microemulsions, Nanoparticles, nanocapsules) or macroemulsions may be encapsulated in hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacrylate) microcapsules, respectively. These techniques are described in Remington's Pharmaceutical Sciences, 16th edition, A. Osal, Ed. (1980).

Compositions used for in vivo administration must be sterile. This is conveniently achieved, for example, by filtration through a sterile filtration membrane. A sustained-release composition may be prepared. A suitable example of a sustained release composition is a semi-permeable matrix of solid hydrophobic polymers bearing antibody variants, which matrices take the form of shaped articles, such as films or microcapsules. Examples of sustained release matrices include polyester, hydrogel (eg, poly(2-hydroxyethyl-methacrylate), or poly(vinyl alcohol)), polylactide (US Pat. No. 3,773,919). ), L-glutamic acid and ethyl-L-glutamate copolymer, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymer (e.g. LUPRON DEPOT ^TM (lactic acid-glycolic acid copolymer and leuprolide acetate))), poly-D-(-)-3-hydroxybutyric acid. Polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid are capable of molecular release of 100 days or more, while certain hydrogels release proteins over a shorter period of time. While the encapsulated antibody remains in the body for a long period of time, it may denature or aggregate as a result of exposure to moisture at 37° C., resulting in loss of biological activity and altered immunogenicity. Rational strategies for stabilization can be devised depending on the mechanisms involved. For example, if the aggregation mechanism is confirmed by the formation of intermolecular SS bonds through thio-disulfide exchange, the sulfhydryl residue is modified, freeze-dried from an acidic solution, moisture content is adjusted, appropriate additives are used, and specific Stabilization can be achieved by developing a suitable polymer matrix composition.

The amount of a therapeutic polypeptide, antibody or fragment thereof that is effective for the treatment of a particular disease or condition depends on the nature of the disease or disease and can be determined by standard clinical techniques. If possible, it is desirable to determine the dose-response curve of the pharmaceutical composition of the present invention in a useful animal model system first in vitro and prior to subsequent clinical examination.

In a preferred embodiment, the aqueous solution of the therapeutic polypeptide, antibody or fragment thereof is administered by subcutaneous injection. Each dose is approximately 0.5 μg to 50 μg/kg body weight, more preferably approximately 3 μg to 30 μg/kg body weight. The dosing schedule for subcutaneous administration varies from once a month to daily, depending on a number of clinical factors, including disease type, disease severity, and patient sensitivity to treatment.

Use of antibody variants

The antibody variant of the present invention can be used as an affinity purification agent. In this process, the antibody is immobilized on a solid phase such as ^SEPHADEX™ resin or filter paper using methods well known to those skilled in the art. The immobilized antibody variant is contacted with a sample containing the target to be purified, and then the support is washed with an appropriate solvent that removes almost all material from the sample except the target to be purified, wherein the target is the immobilized antibody Binds to the variant. Finally, the support is washed with another suitable solvent such as glycine buffer to isolate the target from the antibody variant.

These mutant antibodies can also be used in diagnostic tests to detect the expression of a target of interest in, for example, specific cells, tissues or serum. For diagnostic applications, antibody variants are typically labeled with a detectable moiety. There are a number of labels available for techniques to quantify changes in fluorescence. The chemiluminescent substrate is electrically excited by a chemical reaction and can then be measured (eg using a chemiluminescence analyzer) or emits light that confers energy to the fluorescent receptor. Examples of enzyme labels include luciferase (e.g., firefly luciferase and bacterial luciferase; US Pat. No. 4,737,456), luciferin, 2,3-(dihydrophthalazinedione) (2,3-dihydrophthalazinedione). ), malate dehydrogenase, urease, peroxidase (e.g. horseradish peroxidase (HRPO)), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme , Saccharine oxidase (e.g. glucose oxidase, galactose oxidase, glucose-6-phosphate dehyrogenase), heterocyclic oxidase (e.g., uricase and xanthine oxidase), Lactoferoxidase, microperoxidase, and the like. Techniques for conjugating enzymes to antibodies are described in O'Sullivan et al., 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. Various techniques for achieving this are known to those skilled in the art. For example, antibody variants may be conjugated with biotin, and any one of the above three broad-standard labels may be conjugated with avidin or the like. Biotin specifically binds avidin, so the label can conjugate with antibody variants in this indirect manner. Alternatively, to achieve indirect conjugation of the label and the antibody variant, the antibody variant is conjugated with a small hapten (e.g., digloxin), and one of the different types of labels described above is an anti-hapten antibody variant ( For example, anti-digloxin antibody). Thus, indirect conjugation of the label and antibody variants can be achieved.

In another embodiment of the invention, the antibody variant does not need to be labeled, and its presence can be detected using a labeled antibody that binds to the antibody variant.

The antibody of the present invention can be used in any known test method, for example, a competitive binding test method, a direct and indirect sandwich test method, and an immunoprecipitation test method (Zola, Monoclonal Antibodies: A Manual of Techniques, pp. 147-158). (CRC Press, Inc. 1987).

Competitive binding assays depend on the ability of a labeled standard to compete with a test sample for binding to a limited amount of antibody variants. The amount of target in the test sample is inversely proportional to the amount of reference that binds the antibody. In order to readily determine the amount of criteria to which it binds, antibodies are generally insoluble before and after competition. As a result, the reference and test sample binding to the antibody are conveniently separated from the unbound reference and test sample.

The sandwich assay involves the use of two antibodies, each of which can bind to a different immunogenic moiety, or epitope, or to a protein to be detected. In the sandwich test, the test sample to be analyzed is bound by a first antibody immobilized on a solid support, and then a second antibody binds to the test sample to form an insoluble trilateral complex (see US Pat. No. 4,376,110). . The second antibody can be itself labeled with a detectable moiety (direct sandwich assay), or can be measured using an anti-immunoglobulin antibody labeled with a detectable moiety (indirect sandwich assay). For example, one type of sandwich test is an ELISA test, in which case the detectable part is an enzyme.

For immunohistochemistry, tumor samples are frozen or frozen or embedded in paraffin and fixed with a preservative such as formalin. Antibodies can also be used in in vivo diagnostic tests. In general, antibody variants are labeled with radionucleotides (eg, sup.111 In, sup.99 To, sup.14 C, sup.131 I, sup.3 H, sup.32 P or sup.35 S), and The location of the tumor can be identified using immunoscintigraphy. For example, the high affinity anti-IgE antibodies of the present invention can be used to detect the amount of IgE present in the lungs of asthmatic patients, for example.

The antibodies of the present invention may be provided as a kit, that is, a packaged combination of reactants in a predetermined amount with instructions for performing a diagnostic test. When the antibody variant is labeled with an enzyme, the kit includes the substrate required by the enzyme and a cofactor (eg, a substrate precursor that provides a detectable chromophore or fluorophore). In addition, other additives such as stabilizers, buffers (block buffers or lysis buffers) may be included. The relative amounts of various reactants can be varied widely to provide a concentration in solution of the reactants that substantially optimizes the sensitivity of the assay. In particular, these reactants, including excipients, are generally provided as freeze-dried dry powders, which provide a reactant solution having an appropriate concentration after dissolution.

In vivo use of antibodies

The antibodies of the present invention can be used to treat mammals. In one embodiment, the antibody is administered to a non-human mammal, eg, for the purpose of obtaining preclinical data. Typical non-human mammals to be treated are primates, dogs, cats, rodents, and other mammals for which preclinical studies are conducted. These mammals are either established animal models for diseases treated with the antibodies of the invention, or can be used to study the toxicity of the antibodies of interest. In each of these embodiments, dose escalation studies can be conducted in these mammals. The antibodies or polypeptides of the present invention are administered by any suitable means, including parenteral, subcutaneous, intraperitoneal, intrapulmonary, intranasal and, when necessary for local immunosuppressive treatment, including intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In addition to this, antibody variants are particularly suitably administered in decreasing doses of pulse infusion. Suitably, dosing is provided by infusion, most preferably intravenous or subcutaneous infusion, which depends in part on whether administration is short-term or long-term.

In the prophylaxis or treatment of a disease, the appropriate amount of the antibody or polypeptide is the type and severity of the disease being treated, the progression of the disease, whether the antibody variant is administered for prophylactic or therapeutic purposes, prior therapy, the patient's medical history and the antibody variant. The reaction depends on the judgment of the willingness to participate The high affinity anti-human IgE antibody of the present invention can be appropriately administered to a patient at one time 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 the first candidate dose in patient administration, e.g. by one or more individual administrations, or by continuous infusion. to be. A typical daily dose may be approximately 1 mg/kg to 100 mg/kg or more, depending on the factors described above. In the case of repeated administrations for several days or longer, depending on the condition, treatment continues until the desired suppression of disease symptoms is achieved. However, other dosing regimens may be useful. The progress of treatment is conveniently monitored by routine techniques and tests. Typical dosing regimens for anti-LFA-1 or anti-lCAM-1 antibodies are described in WO 94/04188.

Antibody variant compositions are prepared, formulated and administered in a manner consistent with good medical practice. Factors to be considered in this regard include the specific disease being treated, the specific mammal being treated, the clinical condition of the individual patient, the cause of the disease, the site of delivery of the drug, the method of administration, the dosing regimen, and other factors known to the healthcare professional. do. The “therapeutically effective amount” of the antibody variant administered is determined by these factors and is the minimum amount necessary to prevent, alleviate or treat a disease or condition. Optionally, antibody variants can be prepared in combination with other drugs currently in use to prevent or treat the disease in question. The effective amount of these other drugs depends on the amount of antibody present in the composition, the type of disease or treatment, and other factors described above. In general, they are used in the same dose and route of administration as previously described, or in amounts of approximately 1 to 99% of the dose described above.

Antibodies of the present invention that recognize IgE as a target can be used to treat “IgE-mediated diseases”. These include asthma, allergic rhinitis & conjunctivitis (hay fever), eczema, hives, atopic dermatitis, and food allergies. For example, severe physiological conditions of anaphylactic shock caused by bee stings, snake bites, foods or pharmaceuticals are also within the scope of the present invention.

Antibody epitope genetic mapping

“Epitope” refers to a specific site of an antigen to which B and/or T cells respond. B-cell epitopes can be formed from contiguous amino acids, or non-contiguous amino acids in parallel by tertiary folding of proteins. Epitopes formed from contiguous amino acids are typically retained after exposure to a denaturing solvent, whereas epitopes formed by tertiary folding are typically lost after treatment with a denaturing solvent. Typically, epitopes have at least 3, more usually at least 5 or 8-10 amino acids in a unique spatial form. Antibodies that recognize the same epitope can be identified by simple immunoassays that show the antibody's ability to block binding of other antibodies to the target antigen.

The epitope genomic mapping of the binding site for the high affinity antibody IgE of the present invention includes Western blot analysis for binding, a peptide scan of the CH3 domain of IgE, an alanine scan of the binding region, and the corresponding IgG1. Amino acid substitution from the region, and site direct mutagenesis entails.

The peptide scan of the entire CH3 domain of IgE required 73 overlapping peptides. Each of these peptides performed binding by the labeled anti-IgE antibody of the present invention to identify a specific epitope of IgE that blocks the binding of IgE to high affinity receptors. In the peptide scan, two peptides were identified as potential anti-IgE MAb contact sites in IgE (epitope'A' and epitope'B') (Figure 11). Although epitope'A' and epitope'B' sequences are approximately 80 amino acids apart from the linear sequence, they are located close to each other in the three-dimensional structure of IgE. These two are surface exposed and partially overlap at the FcεRI binding site of IgE, and both peptides have a positively charged residue of Arg and a hydrophobic residue of Pro. 12 illustrates the binding region of epitope B identified by ELISA using a peptide scan.

Determination of which amino acid residues are important for binding of high affinity antibodies within these epitopes was performed by alanine scanning mutagenesis (Cunningham et al., "High-Resolution Epitope Mapping of hGH-Receptor Interactions by Alanine-Scanning. Mutagenesis" Science 244:1081-1085). Alanine was substituted at each residue of epitope A and epitope B, and binding of a high affinity monoclonal antibody was determined (see Example 12 and FIGS. 13 and 14 below).

Active and passive immunization

The invention also relates to pharmaceutical compositions, such as vaccines, containing the peptide immunogenic molecules of the 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 preparing the immunogen of the invention, which process comprises the step of covalently linking at least one peptide of the invention with a moiety capable of inducing an immune response against the peptide.

In addition, the present invention relates to the aforementioned immunogenic peptides used as therapeutic drugs in the treatment of IgE-mediated diseases or diseases, such as allergies or atopic dermatitis, for example.

In addition, the present invention relates to a method of immunizing a mammal from an IgE-mediated disease or disease, such as allergies and atopic dermatitis, the method comprising a therapeutically effective amount of the above immunogenic peptide in need of such treatment. And administering to the patient.

The immunogenic peptides of the present invention are substantially unable to mediate non-cytolytic histamine release, but can induce antibodies with strong serological cross-reactivity with the target amino acid sequence of epitope A and/or epitope B. I can.

The first amount of peptide (eg, approximately 0.2 mg to 5 mg) may be administered intramuscularly, for example, followed by repeated (booster) administrations at 14 to 28 days. Dosage is highly dependent on the patient's age, weight and overall health. Immunization can be “active” or “passive”. In “active” immunization, the subject takes up the immunogenic protein of the invention and the anti-IgE response is actively induced by the subject's immune system.

"Active immunization" is suitable for use in humans, but other mammalian species, such as dogs, can be treated similarly. As used herein, “immunogenic carrier” refers to a substance capable of independently inducing an immunogenic response in a host animal, and these substances are free carboxyl, amino or hydroxyl functional groups in the polypeptide and corresponding functional groups in the immunogenic carrier material. It may be covalently bonded to the polypeptide either directly through the formation of a peptide or ester bond therebetween, or alternatively through a conventional bifunctional linking group, or as a fusion protein. Examples of such immunogenic carriers include albumin such as BSA; Globulin; Serum thyroglobulin; Hemoglobin; Hemocyanin (especially Keyhole Limpet Hemocyanin [KLH]); J. Immunol. 111 1973; 260-268, J. Immunol. 122 1979; 302-308, J. Immun. 98 1967; 893-900, a Am. J. Physiol. 199 1960; Proteins extracted from ascaris as described in 575-578, such as roundworm extract or a purified product thereof; Polylysine; Polyglutamic acid; Lysine-glutamic acid copolymer; And copolymers containing lysine or ornithine. Vaccines are produced using diphtheria toxoid or tetanus toxoid as an immunogenic carrier material (Lepow ML et al., J. of Infectious Diseases 150 1984; 402-406; Coon Beuvery, E. et al., Infection and Immunity 40 1983; 39-45), these toxoid materials can also be used in the present invention. The purified protein derivative (PPD) of tuberculin is particularly suitable for use in “active” immunization strategies, because the derivative does not (1) induce a T-cell response by itself (i.e., it is not practical). As "T-cell hapten") behaves like a fully engineered antigen and is recognized by T-cells; (2) Known as one of the most powerful hapten “carriers” in linked recognition mode; (3) This is because it can be used on humans without additional testing.

Further, the present invention relates to a polynucleotide encoding the peptide of the present invention, a vector containing the polynucleotide, and a cell containing the vector. In addition, active immunization can be achieved by administering a polynucleotide encoding the peptide of the present invention. Vectors suitable for such therapy are known in the art and are, for example, adenovirus vectors.

“Passive” immunization is achieved by administering an anti-IgE antibody of the present invention to a patient suffering from an IgE-mediated disease or disease. These antibodies can be prepared by administering the immunogenic peptide of the present invention to a non-human mammal and collecting the resulting anti-serum. Improved titer can be achieved with repeated injections over a period of time. There are no particular restrictions on the species of mammals that can be used to induce antibodies; In general, it is preferred to use rabbits or guinea pigs, but horses, cats, dogs, goats, pigs, mice, cows and sheep may also be used. Antibodies are collected by collecting blood from the immunized animals 1 to 2 weeks after the final administration, centrifuging the blood, and separating the serum from the blood. The monoclonal antibody can be, for example, a human or murine monoclonal antibody.

When immunizing an individual, the antibody of the invention can be introduced into a mammal, for example by intramuscular injection. However, any form of antibody administration can be used. Any conventional liquid or solid vehicle that is acceptable to the subject and does not cause harmful side effects can be used. The phosphate-buffer (PBS) at physiological pH, e.g., approximately pH 6.8 to 7.2, preferably approximately pH 7.0, is used alone or in combination with a suitable adjuvant, e.g., aluminum hydroxide-based adjuvant. Can be used as.

The following examples are for purposes of illustration and do not limit the present invention.

Example 1: Humanization of anti-IgE murine MAb TES-C21

The sequences of the heavy chain variable region (V _H ) and light chain variable region (V _L ) of the murine mAb TES-C21 were compared to human antibody germ cell line sequences available in public databases. Several criteria, including overall length, similar CDR positions within the framework, overall homology, CDR size, etc., are used when determining the template, as described in step 1 above. Taken together, all these criteria provided results for selecting the optimal human template as shown in the sequence alignment between the TES-C21 MAb heavy and light chain sequences and individual human template sequences shown in Figures 3A and 3B.

In this case, antibodies were designed using one or more human framework templates. The _{human template chosen for the V H} chain was a combination of DP88 (aa residues 1-95) and JH4b (aa residues 103-113) (FIG. 3B ). The _{human template selected for the V L} chain was a combination of L16 (VK subgroup III, aa residues 1-87) and JK4 (as residues 98-107) (Fig. 3A). The framework homology between the murine sequence and the human template was approximately 70% for _{V H} and approximately 74% for _{V L.}

When the template was selected, a Fab library was constructed by DNA synthesis and overlapping PCR as described above and shown in FIG. 2. The library consisted of synthetic TES-C21 CDRs synthesized from selected human templates, DP88/JH4b and L16/JK4, respectively. The complexity of the library was 4096 (= 2 ¹² ). Overlapping nucleotides encoding partial V _H and V _L sequences were synthesized in the range of approximately 63 to 76 nucleotides with 18 to 21 nucleotide overlaps.

PCR amplification of the V _L and V _H genes was performed using a biotinylated forward primer (Genelll) having a specific sequence in the framework region FR1 and an overhanging sequence annealed at the end of the leader sequence, and a rear primer (CK or CH1) from the conserved constant region. ) Was performed under standard PCR conditions. PCR products were purified by agarose gel electrophoresis, or a commercial PCR purification kit to remove non-integrated biotinylated primers and non-specific PCR.

The 5'-phosphorylation of the PCR product was 2 μg PCR product, 1 μl of T4 polynucleotide kinase (10 unit/μl), 2 μl of 10x PNK buffer, 1 μl of the total volume of 20 μl adjusted by _{ddH 2 O.} This was done using 10 mM ATP. 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 _{ddH 2 O for the next step.}

100 μl of streptavidin-coated magnetic beads were washed twice with 200 μl 2x B&W buffer and twice with 200 μl 2x B&W buffer. The phosphorylated PCR product was mixed with beads and incubated for 16 minutes at room temperature (RT) with gentle stirring.

Beads were precipitated and washed twice with 200 μl 2x B&W buffer. Non-biotinylated ssDNA (minus strand) was eluted with 300 μl of freshly prepared 0.15M NaOH for 10 minutes at room temperature with gentle stirring. A second NaOH elution can slightly increase the yield (optional). The eluent was centrifuged to remove any residual beads.

ssDNA was precipitated from the supernatant by adding 1 μl glycogen (10 mg/ml), 1/10 volume 3M NaOAc (pH 5.2), 2.5 volume EtOH. The precipitated ssDNA was washed with 70% EtOH, freeze-dried for 3 minutes, and dissolved in _{20 µl ddH 2 O.} ssDNA was quantified by placing DNA strands on an ethidium bromide (EtBr) agarose plate or _{measuring OD 260.}

Example 2

V as phage-expression vector _H And V _L Cloning of

V _H and V _L were cloned into a phage-expression vector by hybridization mutagenesis. Our dinhwa (uridinylation) mold CJ236 E. coli (E. coli) strain prepared by infecting a - (the expression vector phage TN003) (dut ^{- -} ung) of the basic phage M13-. The following components [200 ng of uridinelated phage vector (8.49 kb); 92 ng phosphorylated single-stranded H chain (489 bases); 100 ng phosphorylated single-stranded L chain (525 bases); 1 [mu]l 1OX annealing buffer; Adjust the volume to 10 [mu]l with ddH ₂ O], which was then annealed by PCR in which the temperature was maintained at 85[deg.] C. for 5 minutes (denaturation), and then ramped to 55[deg.] C. for 1 hour (approximately 8 -In times molar ratio). These samples were cooled on ice.

The annealed product contains the following components: 1.4 μl 10 X synthesis buffer; 0.5 [mu]l T4 DNA ligase (1 unit/[mu]l); 1 µl T4 DNA polymerase (1 unit/µl) was added, followed by incubation on ice for 5 minutes and at 37°C for 1.5 hours. Then, the product was precipitated with ethanol and _{dissolved in 10 μl of ddH 2} O or TE.

DNA was digested with 1 [mu]l Xbal (10 unit/[mu]l) for 2 hours, and heat inactivated at 65[deg.] C. for 20 minutes. The cut DNA was transfected into 50 μl of electro-competent DH1OB cells by electroporation. The resulting phage was titrated by growing on an XL-1 blue bacterial lawn at 37° C. overnight. The clone was sequenced to confirm its composition.

Example 3

Deep well culture for library screening

A. Phage library smear

Phage libraries were diluted in LB medium to achieve the desired number of plaques per plate. The high titer phage was mixed with 200 μl XL-1 B cell culture. 3 ml LB upper layer agar was mixed, poured into LB medium, and allowed to stand at room temperature for 10 minutes. Plates were 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 mM NaCl) was added to each well of a sterile U-bottom 96 well plate. Single phage plaques from the library plate overnight were transferred to the wells with filtered filter tips. The phage elution plate was incubated at 37°C for 1 hour. Plates were stored at 4°C after incubation.

C. Incubation in a deep well plate

XL1B cells from 50 ml culture were added to 2xYT medium at a dilution of 1:100. Cells were grown in a stirrer at 37° C. until _{A 600 was 0.9 to 1.2.}

D. Infection with phage in deep well plates

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

E. Preparation of supernatant for ELISA screening

After incubation, the deep well plate was centrifuged at 3,250 rpm for 20 minutes using a Beckman JA-5.3 plate rotor. For ELISA, 50 µl of the supernatant was collected from each well.

F. Inoculation of 15 ml liquid culture of XL-1 cells

XL-1 was grown on a stirrer (250 rpm) at 37° C. in 2xYT containing 10 μg/ml tetracycline from _{A 600 = 0.9 to 1.2.} To characterize each clone, IPTG was added to a final concentration of 0.5 mM, and 15 ml of the culture was transferred to a 50 ml conical tube. Cells were inoculated with 10 μl phage from a high titer stock solution (titer = ^{˜10 11} pfu/ml) and incubated at 37° C. for 1 hour. These cells were grown overnight at room temperature with stirring.

G. Isolation of Soluble Fab from Periplasm

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

Example 4

Skeletal transformation

There were 12 murine/human wobble residues at these potential key positions in the skeleton. Position 73 in V _H is conserved as a murine residue threonine in a humanization library, because this position has been confirmed to affect binding. However, threonine in VH 73 is a common human residue in the _{human germ cell line V H subgroups 1 and 2.}

The framework residues that distinguish between the TES-C21 sequence and the human template were randomly substituted as described above and then evaluated for their potential effect on target binding and antibody folding. Potential framework residues that can affect binding have been identified. In the case of the present invention to, these were residues at _{V H 12, 27, 43,} 48, 67, 69 and _{V L 1, 3, 4,} 49, 60, 85 in (Kabat notation (number system)) (4) . Subsequently, only 27 and 69 were found to have a significant effect on binding in the _{V H region (clone number 1136-2C).}

The primary screen used was single point ELISA (SPE) using culture medium (see below). The primary screen selected clones that bind to the target molecule of the antibody. Clones giving signals equal to or superior to the parent molecule were selected for subsequent screening. In the secondary screening, individual phages were grown in 15 ml bacterial culture, and periplasmic preparations were used for SPE and ELISA titration assays. Clones retaining high avidity in this test were further characterized. When all selected primary clones were processed, the top 10-15% clones were sequenced and aligned according to sequence. Representatives from each sequence group were compared with each other and the best clone was selected. Sequences from these selected clones were aggregated and the effects of various combinations were evaluated.

The constructed library was subjected to an ELISA screen for improved binding to recombinant human IgE, SE44. A clone having a binding affinity higher than that of the murine TES-C21 Fab was isolated and sequenced. Clone ID #4, 49, 72, 76, 136 were further characterized. ELISA titration curves for clones 4, 49, 72, 78, 136 are shown in Figures 5A and 5B, which suggests that the affinity of these clones is similar to that of the parental TES-C21. These clones compete with murine TES-C21 for binding to human IgE, suggesting that the binding epitope does not change during the humanization process. Humanized Fabs do not bind FcεRI-bound IgE, meaning that when these humanized antibodies are constructed with divalent IgG, they are less likely to cross-link the receptor and induce histamine release. Humanized clone 136 retains 5 murine heavy chain framework residues (= 94.3% human V _H framework homology), with 100% human light chain framework selected by affinity mutation. Inhibition of IgE binding to FcεRI by humanized Fab was demonstrated (FIG. 6 ).

Example 5

Single point ELISA protocol for screening for anti-IgE

Plates were coated with 2 μg/ml anti-human Fd in carbonate coating buffer overnight at 4°C. The coating solution was removed, and the plate was blocked with 200 μl/well 3% BSA/PBS for 1 hour at 37°C. After washing the plate 4x with PBS/0.1% TWEEN (PBST), 50 μl/well Fab sample (i.e., Fab or periplasmic prep secreted from high titer phage and DMB block, or supernatant containing 15 ml prep) Was added. The plate was incubated for 1 hour at room temperature, and then washed 4x with PBST. Then, 50 μl/well of biotinylated SE44 diluted to 0.015 μg/ml in 0.5% BSA/PBS and 0.05% TWEEN was added. Then, the plate was incubated at room temperature for 2 hours and washed 4x with PBST. In 0.5% BSA/PBS and 0.05% TWEEN, 50 µl/well streptavidin HRP 1:2000 dilution was added, and the plate was incubated at room temperature for 1 hour. The plate was washed 6x with PBST. Progress was made by the addition of 50 μl/well TMB substrate (sigma), and then stopped by the addition _{of 50 μl/well 0.2MH 2} SO _4.

Example 6

ELISA titration: anti-IgE

Plates were coated overnight at 4° C. with 0.25 μg/ml (0.1 μg/ml in the case of purified Fab) SE44 in carbonate coating buffer. The coating solution was removed, and the plate was blocked with 200 μl/well 3% BSA/PBS for 1 hour at 37°C.

The plate was washed 4x with PBS/0.1% TWEEN (PBST). A 1:2 dilution was initiated by addition of 50 μl/well Fab (from 15 ml periplasmic prep), followed by serial dilutions 3 times in 0.5% BSA/PBS and 0.05% TWEEN. The plate was incubated for 2 hours at room temperature.

These plates were washed 4x with PBST, and 50 µl/well 1:1000 (0.8 µg/ml) dilution of biotin-yang anti-human Fd in 0.5% BSA/PBS and 0.05% TWEEN was added. The plate was incubated again for 2 hours at room temperature.

After washing 4x with PBST, the plate was added 50 μl/well Neutra-avidin-AP 1:2000 (0.9 μg/ml) in 0.5% BSA/PBS and 0.05% TWEEN, and at room temperature. Incubated for 1 hour.

These plates were washed 4x with PBST. Progress was made by adding 50 μl/well pNPP substrate, then stopped by adding 50 μl/well 3M NaOH. The absorbance of each well was read at 405 nm or 41 Onm.

Example 7

Protocol for affinity purification of M13 phage expressed soluble Fab

A. DAY1

Two 500 ml cultures (2xYT) containing 10 mg/ml tetracycline were inoculated with 5 ml overnight stock solution XL1B _{and grown to A 600} = 0.9 to 1.2 at 37°C. IPTG was added at a concentration of 0.5 mM. Thereafter, the cell culture solution was infected with 200 μl phage per culture solution and incubated at 37° C. for 1 hour while stirring. After infection, these cells were grown overnight at 25° C. with stirring.

B. DAY2

The cells were pelleted in a 250 ml centrifuge tube at 3500 x g for 30 minutes at 4°C. The cell medium was aspirated, and the pellet was resuspended in a total of 12-15 ml of lysis buffer (buffer A + protease inhibitor cocktail).

Buffer A :(1 ℓ)

50mM NaH ₂ PO ₄ 6.9 g NaH ₂ PO ₄ H ₂ O (or 6 g NaH ₂ PO ₄ )

300mM NaCl 17.54 g NaCl

10 mM imidazole 0.68 g imidazole (MW 68.08)

Adjust the pH to 8.0 with NaOH.

Lysis buffer :

25 ml of buffer A is mixed with one tablet of the complete protease inhibitor cocktail (Roche, Basel, Switzerland).

Resuspended cells were transferred to 50 ml conical tubes and lysed with 100 μl 100 mg/ml Iysozyme by inverting the tube several times until the mixture clumped as small droplets (due to dissolution). Cells were sonicated on ice, then 10 μl DNase I (approximately 1000 units) was added and gently shaken at 4° C. for 30 minutes. The debris was centrifuged at 12000 x g for 30 minutes at 4°C using a 50 ml centrifuge tube, and then aggregated into pellets. The supernatant was transferred to a new conical tube and stored at 4°C.

Soluble Fab was purified using Ni-NT agarose (Qiagen, Valencis, CA) according to the manufacturer's protocol. The lysate (Iysate) was mixed with Ni-NTA and added dropwise to the column. The flow through was collected for SDS-PAGE analysis. The column was washed with 20 ml buffer (50 mM NaH ₂ PO ₄ , 300 mM NaCl, 15 mM imidazole, adjust the pH to 8.0 with NaOH), and then _{20 ml containing 50 mM NaH 2} PO ₄ , 300 mM NaCl, 20 mM imidazole Washed with washing solution. Fab was eluted with 6 x 500 μl elution buffer (50mM NaH ₂ PO ₄ , 300mM NaCl, 450mM imidazole, and the pH was adjusted to 8.0 with NaOH) and analyzed by SDS PAGE. Column fractions were stored at 4°C. Column fractions were analyzed by SDS-PAGE, and fractions containing the maximum amount of Fab were selected and dialyzed against PBS at 4°C.

Example 8

Soluble receptor test

A 96-well test plate suitable for ELISA was coated with 0.05 ml 0.5 μg/ml FcεRI alpha-chain receptor coating buffer (50 mM carbonate/bicarbonate, pH 9.6) for 12 hours at 4-8°C. The wells were aspirated, and 250 µl blocking buffer (PBS, 1% BSA, pH 7.2) was added and incubated at 37° C. for 1 hour. In a separate test plate, these samples and reference TES-C21 MAb were titrated to 200 to 0.001 µg/ml at 1:4 dilution with test buffer (0.5% BSA, 0.05% Tween 20, PBS, pH 7.2) and equivalent 100 ng/ml of biotinylated IgE was added, and the plate was incubated at 25° C. for 2-3 hours. The FcεRI-coated wells were washed three times with PBS and 0.05% TWEEN 20, and 50 µl was transferred from the sample well and incubated at 25° C. for 30 minutes while stirring. 50 μl/well of 1 mg/ml streptavidin-HRP diluted 1:2000 in the test buffer was incubated for 30 minutes while stirring, and then the plate was washed as described above. 50 μl/well of TMB substrate was added and color was developed. The reaction was stopped by adding an equivalent amount of 0.2 MH ₂ SO ₄ , and absorbance was measured at 450 nm.

Example 9

Binding of antibodies to IgE-loaded FcεRI

Antibody binding to human IgE bound to the alpha-subunit of FcεRI was determined by pre-incubation with 10 μg/ml human IgE at 4° C. for 30 minutes. Plates were washed 3 times and then incubated for 1 hour with variable concentrations of murine anti-human IgE MAb E-10-10 or humanized Fab variants. The binding of Fab was sequentially detected with biotin-labeled anti-human Fd antibody and SA-HRP. Murine MAb E-10-10 was detected with goat anti-murine Ig Fc HRP-conjugated Ab.

Example 10

Clone characterization

Each candidate was analyzed for binding affinity, and the positive clone was sequenced. Antibody variants bearing beneficial mutations in the CDR regions that increase binding affinity were further characterized. Biacore analysis on examination; Inhibition of binding of IgE to receptors; Cross-linking of receptor bound IgE was included.

I created a library of variants. The amino acid sequences of various CDRs showing improved affinity are shown in Table 1. 7 shows high affinity candidates with a combination of substitutions.

19 heavy chain variants are shown in Figure 9, and 35 light chain variants are shown in Figure 8. The three candidates were further characterized for binding affinity, which are shown in Table 2.

Binding affinity Mab K _D Increase in binding affinity (fold) TES-C21 614±200 pM MAb 1 (CL-5A) 0.158 pM 3886 MAb 2 (CL-2C) 1.47±0.5 pM 417 MAb 3 (CL-5I) 3.2±2.2 pM 191

Example 11

Expression and purification of anti-IgE antibody and HRP-conjugation

High affinity MAb antibodies were produced. For the production of circular anti-IgE MAb, the heavy and light chain variable regions were amplified by PCR from a phage vector template, and subcloned into H-chain and L-chain expression vectors separately under the expression of the CMV promoter. Six complete antibody clones were constructed (FIGS. 10A-F). Appropriate heavy and light chain plasmids were co-transfected into the mouse myeloma cell line NSO using electroporation by techniques well known in the art (Liou et al. J Immunol. 143(12):3967-75) (1989)). Antibodies were purified from the supernatant of a single stable cell line using protein A-Sepharose (Pharmacia). The concentration of the antibody was determined at 280 nm using a spectrophotometer and an FCA assay (IDEXX).

The purified antibody was conjugated with horseradish peroxidase (HRP) using a peroxidase conjugation kit (Zymed Labs, San Francisco, CA) according to the manufacturer's protocol. The titer of each conjugated anti-IgE MAb was determined using ELISA with a plate coated with monoclonal human IgE (SE44).

The cultures below are from the American Type Culture Collection, 10801 University Boulevard, Manassas Va. 201 10-2209 deposited in USA (ATCC):

The deposit was carried out under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure and the Regulations thereunder. This guarantees the maintenance of the viable culture for 30 years from the date of deposit. Microorganisms are related to U.S. After the publication of the patent publication, it is available from ATCC under the provisions of the Budapest Treaty, which guarantees the permanent and unlimited availability of the offspring of culture to the general public. The applicant of the present invention has agreed that if a deposited culture dies, is lost or destroyed in the process of being cultivated under appropriate conditions, it is replaced with a viable specimen of the same culture upon receipt of notification. The availability of the deposited strain is not regarded as an acceptance to practice the present invention contrary to the rights granted by any governmental agency under its patent law.

It is believed that the above specification is sufficient to enable a person skilled in the art to practice the present invention. The invention is not limited to the deposited cultures, as these deposited embodiments illustrate one aspect of the invention, and any culture that is functionally equivalent falls within the scope of the invention. The deposit of substances in the present invention acknowledges that the specification of the present invention is not sufficient to carry out any aspect of the present invention, including the optimum mode, or that the claims may be limited to the specific examples represented by the specification of the present invention Is not considered to be. Indeed, various modifications of the present invention other than those shown and described herein are apparent to those skilled in the art from the detailed description and fall within the scope of the appended claims.

Example 12

Genetic Mapping of High Affinity Binding Epitopes of Human IgE

A. Peptide Synthesis and Anti-IgE Binding Test

It has been shown that IgE binds to the receptor through the _{C H 3 domain.} Since the HA anti-IgE antibody of the present invention very effectively blocks the binding of IgE to its receptor, a genetic map of the epitope was created using a peptide covering the _{entire C H 3 domain.} First, two V5-labeled peptides were prepared, one containing the complete constant region of human IgE and the other peptide _{containing only the C H} 2-C _H 3 region of human IgE. These two peptides were expressed by in vitro transcription-translation and used in Western blot assay to detect HA anti-IgE MAb binding. Both CL-2C and CL-5I MAbs were able to bind to circular human IgE and these peptides.

In order to map the epitope more specifically, 73 overlapping peptides containing amino acid residues 141 to 368 of human IgE having the _{entire C H 3 domain were synthesized.} Each peptide consisted of 12 amino acid residues with 3 amino acid overlaps with the 3'end of the previous peptide. The SPOT membrane was synthesized from the amino acid fluorenylmethoxycarbonyl (Fmoc) in the cellulose membrane. These membranes were washed in methanol and then washed in TBS (pH 7.5) 3X for 10 minutes. After overnight incubation in blocking solution (5% milk or 3% BSA in TBS), HRP-labeled anti-IgE MAb diluted in blocking solution was incubated with the membrane for 3 hours. After washing 3X for 15 minutes in TBS-TWEEN using SuperSignal HRP substrate (Pierce), IgE reactivity (IgE reactivity) was measured by chemiluminescence exposure of a BioMax MS film (Kodak) for a desired time.

Results from this experiment indicate that HA anti-IgE MAb _{binds to two regions in the C H} 3 portion of IgE, which is represented by the following two peptide sequences: NPRGVSAYLSRP (epitope “A”) and HPHLPRALMRST (epitope "B") (Fig. 12). Binding to epitope A was several times weaker than binding to epitope B.

B. Alanine scan mutagenesis

An alanine scan with substitution of amino acids in these peptides to those found in IgG1 was performed to determine which amino acids are critically involved in the binding of HA anti-IgE MAb to these peptides. Amino acids identified as important for HA anti-IgE MAb binding were replaced in the ε chain of IgE using an in vitro mutagenesis strategy. Other existing peptides spanning the Cε2 and Cε3 regions were also used in this study (Figs. 13 and 14).

The EU numbering scheme for human IgE amino acid residues was used. Polymerase chain reaction (PCR) was used to amplify the truncated form of IgE Fc having only the entire Fc region of IgE and the CH2-CH3 domain. DNA products were directly cloned into pcDNA3 expression vectors (Invitrogene, Carlsbad, CA) using TOPO cloning (Invitrogene, Carlsbad, CA).

Mutagenesis in IgE-Fc was performed using overlapping PCR (Ho et al., 1989). The DNA product was purified by agarose gel electrophoresis, digested with an appropriate restriction enzyme, and subcloned into the pcDNA3 expression vector. In each variant construct, the PCR amplified region was completely sequenced from both strands of DNA using the dideoxynucleotide method. Recombinant human IgE Fc and its variants were expressed using a reticulocyte lysate-based in vitro transcription and translation binding system (Promega, Madison, Wl).

The lysate from the in vitro transcription and translation binding system (10 μl reaction mixture) was subjected to SDS-PAGE (12%), and then transferred to a nitrocellulose membrane. The membrane was blocked with 5% dry milk dissolved in Tris-buffer (TBS), and then stained with a primary antibody, anti-IgE MAb. Specific reactive bands were detected using goat anti-human IgG Fc conjugated to horseradish peroxidase (Jackson Labs, Bar Harbor, Maine), and immunoreactive bands were visualized with SuperSignal Western blotting detection kit (Pierce). Anti-V5 antibodies were used as positive controls to detect V5 tags introduced at the C-terminus of these peptides. Western blot using anti-V5 antibody confirmed that all these peptides were expressed at approximately equal levels. Interestingly, the HA anti-IgE MAb was able to bind to a peptide carrying a mutation in epitope'A', but not to a peptide carrying a mutation in epitope'B', indicating that the second site is more important for binding. Is implied (Fig. 15).

Example 13

Active Immunization of Transgenic Mice Using Immunogenic Peptides of Epitope B

The active production of antibodies against human immunogenic peptides of epitope B was demonstrated using transgenic mice that structurally express human IgE. Two fusion peptides, each comprising an immunogenic peptide of the present invention, a cysteine residue, and KLH, were chemically synthesized. The sequence of peptide 1 is as follows:

(KLH-Cys)-Leu Pro Arg Ala Leu Met Arg Ser Thr;

The sequence of peptide 2 is as follows:

Leu Pro Arg Ala Leu Met Arg Ser Thr-(Cys-KLH)

These transgenic mice were injected subcutaneously with 20 µg of immunogenic peptide contained in complete Freund's adjuvant (Difco Laboratories, Detroit, MI) in 200 µl of PBS (pH 7.4). At intervals of two weeks, these mice were injected subcutaneously twice with 20 μg of peptide immunogen contained in an incomplete Freund adjuvant. Two weeks later, 3 days before sacrifice, these mice were again intraperitoneally injected with 20 µg of the same immunogen contained in PBS. Serum was collected and examined for the presence of anti-IgE antibodies specific for epitope B. As shown in Fig. 16, the peptide induced anti-IgE antibodies in these transgenic mice.

One skilled in the art will recognize or be able to ascertain, using routine experimentation, a number of equivalents to the specific embodiments of the invention described herein. These equivalents are included in the claims below.

American Type Culture Collection PTA-5678 20031203 American Type Culture Collection PTA-5679 20031203 American Type Culture Collection PTA-5680 20031203

SEQUENCE LISTING <110> TANOX, INC. SINGH, Sanjaya HUANG, Danyang FUNG, Sek Chung <120> Identification of Unique, High Affinity IgE Epitopes <130> 12279-175-187 <150> PCT/US04/02892 <151> 2004-02-02 <150> PCT/US04/02894 <151> 2004-02-02 <150> US60/444,229 <151> 2003-02-01 <160> 78 <170> PatentIn version 3.2 <210> 1 <211> 107 <212> PRT <213> Murine <220> <221> misc_feature <223> TES-C21 LIGHT CHAIN <400> 1 Asp Ile Leu Leu Thr Gln Ser Pro Ala Ile Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Ser Ile Gly Thr Asn 20 25 30 Ile His Trp Tyr Gln Gln Arg Thr Asp Gly Ser Pro Arg Leu Leu Ile 35 40 45 Lys Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Asn Ile Asn Ser Val Glu Ser 65 70 75 80 Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Ser Asp Ser Trp Pro Thr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 2 <211> 107 <212> PRT <213> Human <220> <221> misc_feature <223> L16/-JK4 human light chain consensus sequence template <400> 2 Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Asn 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asn Asn Trp Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 3 <211> 123 <212> PRT <213> MURINE <220> <221> misc_feature <223> TES-C21 Heavy Chain <400> 3 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Met Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Thr Thr Gly Tyr Thr Phe Ser Met Tyr 20 25 30 Trp Leu Glu Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu Trp Val 35 40 45 Gly Glu Ile Ser Pro Gly Thr Phe Thr Thr Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Ala Lys Ala Thr Phe Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr 65 70 75 80 Leu Gln Leu Ser Gly Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala Arg Phe Ser His Phe Ser Gly Ser Asn Tyr Asp Tyr Phe Asp Tyr 100 105 110 Trp Gly Gln Gly Thr Ser Leu Thr Val Ser Ser 115 120 <210> 4 <211> 113 <212> PRT <213> Human <220> <221> misc_feature <223> DP88/JH4b human heavy chain consensus sequence template <400> 4 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30 Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Tyr Phe Asp Tyr Leu Val Gln Gly Thr Ser Leu Thr Val Ser 100 105 110 Ser <210> 5 <211> 11 <212> PRT <213> ARTIFICIAL <220> <223> TES-C21 CDRL1 SEQUENCE (TABLE 1) <400> 5 Arg Ala Ser Gln Ser Ile Gly Thr Asn Ile His 1 5 10 <210> 6 <211> 11 <212> PRT <213> ARTIFICIAL <220> <223> CDRL1 VARIANT SEQUENCE #1 (TABLE 1) <400> 6 Arg Ala Ser Arg Ser Ile Gly Thr Asn Ile His 1 5 10 <210> 7 <211> 11 <212> PRT <213> ARTIFICIAL <220> <223> CDRL1 VARIANT SEQUENCE #2 (TABLE 1) <400> 7 Arg Ala Ser Gln Arg Ile Gly Thr Asn Ile His 1 5 10 <210> 8 <211> 7 <212> PRT <213> ARTIFICIAL <220> <223> TES-C21 CDRL2 SEQUENCE (TABLE 1) <400> 8 Tyr Ala Ser Glu Ser Ile Ser 1 5 <210> 9 <211> 7 <212> PRT <213> ARTIFICIAL <220> <223> CDRL2 VARIANT #1 (TABLE 1) <400> 9 Tyr Ala Tyr Glu Ser Ile Ser 1 5 <210> 10 <211> 7 <212> PRT <213> ARTIFICIAL <220> <223> CDRL2 VARIANT #2 (TABLE 1) <400> 10 Tyr Ala Ser Glu Ser Ile Tyr 1 5 <210> 11 <211> 7 <212> PRT <213> ARTIFICIAL <220> <223> CDRL2 VARIANT #3 (TABLE 1) <400> 11 Tyr Ala Ser Glu Ser Asp Ser 1 5 <210> 12 <211> 7 <212> PRT <213> ARTIFICIAL <220> <223> CDRL2 VARIANT #4 (TABLE 1) <400> 12 Tyr Ala Ser Glu Ser Glu Ser 1 5 <210> 13 <211> 9 <212> PRT <213> ARTIFICIAL <220> <223> TES-C21 CDRL3 (TABLE 1) <400> 13 Gln Gln Ser Asp Ser Trp Pro Thr Thr 1 5 <210> 14 <211> 9 <212> PRT <213> ARTIFICIAL <220> <223> CDRL3 VARIANT (TABLE) <400> 14 Ala Ala Ser Trp Ser Trp Pro Thr Thr 1 5 <210> 15 <211> 5 <212> PRT <213> ARTIFICIAL <220> <223> TES-C21 CDRH1 <400> 15 Met Tyr Trp Leu Glu 1 5 <210> 16 <211> 5 <212> PRT <213> ARTIFICIAL <220> <223> CDRH1 VARIANT #1 (TABLE 1) <400> 16 Trp Tyr Trp Leu Glu 1 5 <210> 17 <211> 5 <212> PRT <213> ARTIFICIAL <220> <223> CDRH1 #2 (TABLE 1) <400> 17 Tyr Tyr Trp Leu Glu 1 5 <210> 18 <211> 17 <212> PRT <213> ARTIFICIAL <220> <223> TES-C21 CDRH2 (TABLE 1) <400> 18 Glu Ile Ser Pro Gly Thr Phe Thr Thr Asn Tyr Asn Glu Lys Phe Lys 1 5 10 15 Ala <210> 19 <211> 17 <212> PRT <213> ARTIFICIAL <220> <223> CDRH2 VARIANT #1 (TABLE 1) <400> 19 Glu Ile Glu Pro Gly Thr Phe Thr Thr Asn Tyr Asn Glu Lys Phe Lys 1 5 10 15 Ala <210> 20 <211> 17 <212> PRT <213> ARTIFICIAL <220> <223> CDRH2 VARIANT #2 (TABLE 1) <400> 20 Glu Ile Asp Pro Gly Thr Phe Thr Thr Asn Tyr Asn Glu Lys Phe Lys 1 5 10 15 Ala <210> 21 <211> 17 <212> PRT <213> ARTIFICIAL <220> <223> CDRH2 VARIANT #3 (TABLE 1) <400> 21 Glu Ile Ser Pro Asp Thr Phe Thr Thr Asn Tyr Asn Glu Lys Phe Lys 1 5 10 15 Ala <210> 22 <211> 17 <212> PRT <213> ARTIFICIAL <220> <223> CDRH2 VARIANT #4 (TABLE 1) <400> 22 Glu Ile Ser Pro Glu Thr Phe Thr Thr Asn Tyr Asn Glu Lys Phe Lys 1 5 10 15 Ala <210> 23 <211> 17 <212> PRT <213> ARTIFICIAL <220> <223> CDRH2 VARIANT #5 (TABLE 1) <400> 23 Glu Ile Ser Pro Gly Thr Phe Glu Thr Asn Tyr Asn Glu Lys Phe Lys 1 5 10 15 Ala <210> 24 <211> 17 <212> PRT <213> ARTIFICIAL <220> <223> CDRH2 VARIANT #6 (TABLE 1) <400> 24 Glu Ile Glu Pro Gly Thr Phe Glu Thr Asn Tyr Asn Glu Lys Phe Lys 1 5 10 15 Ala <210> 25 <211> 17 <212> PRT <213> ARTIFICIAL <220> <223> CDRH2 VARIANT #7 (TABLE 1) <400> 25 Glu Ile Asp Pro Gly Thr Phe Glu Thr Asn Tyr Asn Glu Lys Phe Lys 1 5 10 15 Ala <210> 26 <211> 14 <212> PRT <213> ARTIFICIAL <220> <223> TES-C21 CDRH3 (TABLE 1) <400> 26 Phe Ser His Phe Ser Gly Ser Asn Tyr Asp Tyr Phe Asp Tyr 1 5 10 <210> 27 <211> 14 <212> PRT <213> ARTIFICIAL <220> <223> CDRH3 VARIANT #1 (TABLE 1) <400> 27 Phe Ser His Phe Ser Gly Met Asn Tyr Asp Tyr Phe Asp Tyr 1 5 10 <210> 28 <211> 14 <212> PRT <213> ARTIFICIAL <220> <223> CDRH3 VARIANT #2 (TABLE 1) <400> 28 Phe Ser His Phe Ser Gly Gln Asn Tyr Asp Tyr Phe Asp Tyr 1 5 10 <210> 29 <211> 14 <212> PRT <213> ARTIFICIAL <220> <223> CDRH3 VARIANT #3 (TABLE 1) <400> 29 Phe Ser His Phe Thr Gly Ser Asn Tyr Asp Tyr Phe Asp Tyr 1 5 10 <210> 30 <211> 23 <212> PRT <213> ARTIFICIAL <220> <223> FRL1 VARIANT 136 <400> 30 Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys 20 <210> 31 <211> 23 <212> PRT <213> ARTIFICIAL <220> <223> FRL1 VARIANT 1 <400> 31 Asp Ile Leu Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys 20 <210> 32 <211> 23 <212> PRT <213> ARTIFICIAL <220> <223> FRL1 VARIANT 2 <400> 32 Asp Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys 20 <210> 33 <211> 23 <212> PRT <213> ARTIFICIAL <220> <223> FRL1 VARIANT 4 <400> 33 Asp Ile Leu Leu Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys 20 <210> 34 <211> 23 <212> PRT <213> ARTIFICIAL <220> <223> FRL1 VARIANT 13 <400> 34 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys 20 <210> 35 <211> 23 <212> PRT <213> ARTIFICIAL <220> <223> FRL1 VARIANT 18 <400> 35 Glu Ile Leu Leu Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys 20 <210> 36 <211> 23 <212> PRT <213> ARTIFICIAL <220> <223> FRL1 VARIANT 25 <400> 36 Asp Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys 20 <210> 37 <211> 23 <212> PRT <213> ARTIFICIAL <220> <223> FRL1 VARIANT 27 <400> 37 Glu Ile Leu Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys 20 <210> 38 <211> 15 <212> PRT <213> ARTIFICIAL <220> <223> FRL2 VARIANT 136 <400> 38 Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr 1 5 10 15 <210> 39 <211> 15 <212> PRT <213> ARTIFICIAL <220> <223> FRL2 VARIANT 1 <400> 39 Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Lys 1 5 10 15 <210> 40 <211> 32 <212> PRT <213> ARTIFICIAL <220> <223> FRL3 VARIANT 136 <400> 40 Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr 1 5 10 15 Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp Phe Ala Val Tyr Tyr Cys 20 25 30 <210> 41 <211> 32 <212> PRT <213> ARTIFICIAL <220> <223> FRL3 VARIANT 1 <400> 41 Gly Ile Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr 1 5 10 15 Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp Phe Ala Val Tyr Tyr Cys 20 25 30 <210> 42 <211> 32 <212> PRT <213> ARTIFICIAL <220> <223> FRL3 VARIANT 13 <400> 42 Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr 1 5 10 15 Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp Phe Ala Asp Tyr Tyr Cys 20 25 30 <210> 43 <211> 32 <212> PRT <213> ARTIFICIAL <220> <223> FRL3 VARIANT 18 <400> 43 Gly Ile Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr 1 5 10 15 Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp Phe Ala Asp Tyr Tyr Cys 20 25 30 <210> 44 <211> 10 <212> PRT <213> ARTIFICIAL <220> <223> FRL4 VARIANT <400> 44 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 1 5 10 <210> 45 <211> 30 <212> PRT <213> ARTIFICIAL <220> <223> FRH1 VARIANT 136 <400> 45 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Met Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser 20 25 30 <210> 46 <211> 30 <212> PRT <213> ARTIFICIAL <220> <223> FRH1 VARIANT 2 <400> 46 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser 20 25 30 <210> 47 <211> 14 <212> PRT <213> ARTIFICIAL <220> <223> FRH2 VARIANT 136 <400> 47 Trp Val Arg Gln Ala Pro Gly His Gly Leu Glu Trp Met Gly 1 5 10 <210> 48 <211> 14 <212> PRT <213> ARTIFICIAL <220> <223> FRH2 VARIANT 2 <400> 48 Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly 1 5 10 <210> 49 <211> 14 <212> PRT <213> ARTIFICIAL <220> <223> FRH2 VARIANT 8 <400> 49 Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Val Gly 1 5 10 <210> 50 <211> 14 <212> PRT <213> ARTIFICIAL <220> <223> FRH2 VARIANT 21 <400> 50 Trp Val Arg Gln Ala Pro Gly His Gly Leu Glu Trp Val Gly 1 5 10 <210> 51 <211> 32 <212> PRT <213> ARTIFICIAL <220> <223> FRH3 VARIANT 136 <400> 51 Arg Val Thr Phe Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr Met Glu 1 5 10 15 Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 20 25 30 <210> 52 <211> 32 <212> PRT <213> ARTIFICIAL <220> <223> FRH3 VARIANT 1 <400> 52 Arg Ala Thr Phe Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr Met Glu 1 5 10 15 Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 20 25 30 <210> 53 <211> 32 <212> PRT <213> ARTIFICIAL <220> <223> FRH3 VARIANT 43 <400> 53 Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr Met Glu 1 5 10 15 Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 20 25 30 <210> 54 <211> 32 <212> PRT <213> ARTIFICIAL <220> <223> FRH3 VARIANT 103 <400> 54 Arg Ala Thr Ile Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr Met Glu 1 5 10 15 Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg 20 25 30 <210> 55 <211> 11 <212> PRT <213> ARTIFICIAL <220> <223> FRH4 VARIANT 136 <400> 55 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 <210> 56 <211> 19 <212> PRT <213> Bacteriophage M13mp18 <220> <221> misc_feature <223> Gene III signal sequence <400> 56 Met Glu Trp Ser Gly Val Phe Met Phe Leu Leu Ser Val Thr Ala Gly 1 5 10 15 Val His Ser <210> 57 <211> 107 <212> PRT <213> ARTIFICIAL <220> <223> LIGHT CHAIN VARIABLE REGION OF CLONE 136 <400> 57 Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Gly Thr Asn 20 25 30 Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Asp Ser Trp Pro Thr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 58 <211> 106 <212> PRT <213> ARTIFICIAL <220> <223> LIGHT CHAIN CONSTANT REGION OF CLONE 136 <400> 58 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 1 5 10 15 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 20 25 30 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 35 40 45 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 50 55 60 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 65 70 75 80 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 85 90 95 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 59 <211> 123 <212> PRT <213> ARTIFICIAL <220> <223> HEAVY CHAIN VARIABLE REGION OF CLONE 136 <400> 59 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Met Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Met Tyr 20 25 30 Trp Leu Glu Trp Val Arg Gln Ala Pro Gly His Gly Leu Glu Trp Met 35 40 45 Gly Glu Ile Ser Pro Gly Thr Phe Thr Thr Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Ala Arg Val Thr Phe Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Phe Ser His Phe Ser Gly Ser Asn Tyr Asp Tyr Phe Asp Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 60 <211> 330 <212> PRT <213> ARTIFICIAL <220> <223> CONSTANT REGION OF HUMAN IgG1 <400> 60 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 61 <211> 107 <212> PRT <213> ARTIFICIAL <220> <223> LIGHT CHAIN VARIABLE REGION OF CLONE CL-2C <400> 61 Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Gly Thr Asn 20 25 30 Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Trp Ser Trp Pro Thr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 62 <211> 123 <212> PRT <213> ARTIFICIAL <220> <223> HEAVY CHAIN VARIABLE REGION OF CLONE CL-2C <400> 62 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Met Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Trp Tyr 20 25 30 Trp Leu Glu Trp Val Arg Gln Ala Pro Gly His Gly Leu Glu Trp Met 35 40 45 Gly Glu Ile Asp Pro Gly Thr Phe Thr Thr Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Ala Arg Val Thr Phe Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Phe Ser His Phe Ser Gly Ser Asn Tyr Asp Tyr Phe Asp Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 63 <211> 107 <212> PRT <213> ARTIFICIAL <220> <223> LIGHT CHAIN VARIABLE REGION OF CLONE CL-5I <400> 63 Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Gly Thr Asn 20 25 30 Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Trp Ser Trp Pro Thr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 64 <211> 123 <212> PRT <213> ARTIFICIAL <220> <223> HEAVY CHAIN VARIABLE REGION OF CLONE CL-5I <400> 64 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Met Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Met Tyr 20 25 30 Trp Leu Glu Trp Val Arg Gln Ala Pro Gly His Gly Leu Glu Trp Met 35 40 45 Gly Glu Ile Asp Pro Gly Thr Phe Glu Thr Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Ala Arg Val Thr Phe Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Phe Ser His Phe Ser Gly Ser Asn Tyr Asp Tyr Phe Asp Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 65 <211> 107 <212> PRT <213> ARTIFICIAL <220> <223> LIGHT CHAIN VARIABLE REGION OF CLONE CL-5A <400> 65 Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Gly Thr Asn 20 25 30 Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Trp Ser Trp Pro Thr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 66 <211> 123 <212> PRT <213> ARTIFICIAL <220> <223> HEAVY CHAIN VARIABLE REGION OF CLONE CL-5A <400> 66 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Met Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Trp Tyr 20 25 30 Trp Leu Glu Trp Val Arg Gln Ala Pro Gly His Gly Leu Glu Trp Met 35 40 45 Gly Glu Ile Glu Pro Gly Thr Glu Thr Thr Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Ala Arg Val Thr Phe Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Phe Ser His Phe Ser Gly Ser Asn Tyr Asp Tyr Phe Asp Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 67 <211> 107 <212> PRT <213> ARTIFICIAL <220> <223> LIGHT CHAIN VARIABLE REGION OF CLONE CL-2B <400> 67 Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Gly Thr Asn 20 25 30 Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Trp Ser Trp Pro Thr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 68 <211> 123 <212> PRT <213> ARTIFICIAL <220> <223> HEAVY CHAIN VARIABLE REGION OF CLONE CL-2B <400> 68 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Met Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Tyr Tyr 20 25 30 Trp Leu Glu Trp Val Arg Gln Ala Pro Gly His Gly Leu Glu Trp Met 35 40 45 Gly Glu Ile Asp Pro Gly Thr Phe Thr Thr Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Ala Arg Val Thr Phe Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Phe Ser His Phe Ser Gly Ser Asn Tyr Asp Tyr Phe Asp Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 69 <211> 107 <212> PRT <213> ARTIFICIAL <220> <223> LIGHT CHAIN VARIABLE REGION OF CLONE CL-1136-2C <400> 69 Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Gly Thr Asn 20 25 30 Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Trp Ser Trp Pro Thr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 70 <211> 123 <212> PRT <213> ARTIFICIAL <220> <223> HEAVY CHAIN VARIABLE REGION OF CLONE CL-1136-2C <400> 70 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Met Tyr 20 25 30 Trp Leu Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Glu Ile Ser Pro Gly Thr Phe Thr Thr Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Ala Arg Val Thr Phe Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Phe Ser His Phe Ser Gly Ser Asn Tyr Asp Tyr Phe Asp Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 71 <211> 108 <212> PRT <213> Homo sapiens <400> 71 Asp Ser Asn Pro Arg Gly Val Ser Ala Tyr Leu Ser Arg Pro Ser Pro 1 5 10 15 Phe Asp Leu Phe Ile Arg Lys Ser Pro Thr Ile Thr Cys Leu Val Val 20 25 30 Asp Leu Ala Pro Ser Lys Gly Thr Val Asn Leu Thr Trp Ser Arg Ala 35 40 45 Ser Gly Lys Pro Val Asn His Ser Thr Arg Lys Glu Glu Lys Gln Arg 50 55 60 Asn Gly Thr Leu Thr Val Thr Ser Thr Leu Pro Val Gly Thr Arg Asp 65 70 75 80 Trp Ile Glu Gly Glu Thr Tyr Gln Cys Arg Val Thr His Pro His Leu 85 90 95 Pro Arg Ala Leu Met Arg Ser Thr Thr Lys Thr Ser 100 105 <210> 72 <211> 12 <212> PRT <213> ARTIFICIAL <220> <223> PEPTIDE DERIVED FROM CH3 DOMAIN OF IGE <220> <221> misc_feature <222> (7)..(7) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (9)..(10) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (12)..(12) <223> Xaa can be any naturally occurring amino acid <400> 72 Asn Pro Arg Gly Val Ser Xaa Tyr Xaa Xaa Arg Xaa 1 5 10 <210> 73 <211> 12 <212> PRT <213> ARTIFICIAL <220> <223> PEPTIDE DERIVED FROM CH3 DOMAIN OF IGE <400> 73 Asn Pro Arg Gly Val Ser Ala Tyr Leu Ser Arg Pro 1 5 10 <210> 74 <211> 9 <212> PRT <213> ARTIFICIAL <220> <223> PEPTIDE DERIVED FROM CH3 DOMAIN OF IGE <220> <221> misc_feature <222> (6)..(6) <223> Xaa can be any naturally occurring amino acid <220> <221> misc_feature <222> (9)..(9) <223> Xaa can be any naturally occurring amino acid <400> 74 Leu Pro Arg Ala Leu Xaa Arg Ser Xaa 1 5 <210> 75 <211> 9 <212> PRT <213> ARTIFICIAL <220> <223> PEPTIDE DERIVED FROM CH3 DOMAIN OF IGE <400> 75 Leu Pro Arg Ala Leu Met Arg Ser Thr 1 5 <210> 76 <211> 12 <212> PRT <213> ARTIFICIAL <220> <223> PEPTIDE DERIVED FROM CH3 DOMAIN OF IGE <400> 76 His Pro His Leu Pro Arg Ala Leu Met Arg Ser Thr 1 5 10 <210> 77 <211> 12 <212> PRT <213> ARTIFICIAL <220> <223> PEPTIDE DERIVED FROM CH3 DOMAIN OF IGE <400> 77 Leu Pro Arg Ala Leu Met Arg Ser Thr Thr Lys Thr 1 5 10 <210> 78 <211> 9 <212> PRT <213> Artificial <220> <223> CDRL3 VARIANT SEQUENCE #2 <400> 78 Gln Gln Ser Trp Ser Trp Pro Thr Thr 1 5

Claims (13)

CDRL1, CDRL2, and CDRL3, and a variable heavy chain region comprising CDRH1, CDRH2 and CDRH3, or an antigen-binding fragment thereof, that specifically binds IgE, CDRL1 is selected from the group consisting of SEQ ID NO: 5, CDRL2 is composed of SEQ ID NO: 8, CDRL3 is composed of the amino acid sequence consisting of SEQ ID NO: 78, CDRH1 is composed of SEQ ID NO: 16, CDRH2 is composed of SEQ ID NO: And CDRH3 is comprised of SEQ ID NO: 26. 2. The antibody of claim 1, comprising an invariant light chain constant region and an invariant heavy chain region. The antibody of claim 2, wherein the constant light chain region has the amino acid sequence set forth in SEQ ID NO: 58 and the constant heavy chain region has the amino acid sequence set forth in SEQ ID NO: 60. A composition for treating a disorder associated with abnormal IgE, comprising an antibody of any one of claims 1 to 3 and a pharmaceutically acceptable carrier, said disorder being selected from the group consisting of asthma, allergic rhinitis, eczema, urticaria, Dermatitis or food allergy. The antibody of any one of claims 1 to 3, wherein the antibody is further linked to a label. A diagnostic kit comprising an antibody of any one of claims 1 to 3 for use in an in vitro method of determining the level of IgE in tissue obtained from an individual. A nucleic acid encoding an antibody according to any one of claims 1 to 3. 9. A vector comprising a nucleic acid encoding the antibody of claim 7. 9. A cell comprising the vector of claim 8. A method for measuring the level of IgE in an individual other than a human, the method comprising contacting a sample isolated from an individual with an antibody of any one of claims 1 to 3, wherein the sample comprises IgE molecules ; And comparing the control level of the antibody with the control sample of the control individual, wherein higher or lower level antibody retention by the sample of the individual relative to the control sample indicates that the individual is Lt; RTI ID = 0.0 &gt; IgE &lt; / RTI &gt; molecule. A method for detecting an IgE-mediated disease in a subject other than a human, the method comprising contacting a sample isolated from an individual with an antibody of any one of claims 1 to 3, wherein the sample comprises an IgE molecule Includes; And determining the level of retention of the antibody by the sample of the individual compared to a control sample of the control individual wherein higher or lower levels of antibody retention by the sample of the individual relative to the control sample is indicative that the individual is an IgE- RTI ID = 0.0 &gt; a &lt; / RTI &gt; mediated disease. 12. The method of claim 11, wherein the IgE-mediated disease is asthma, allergic rhinitis, eczema, urticaria, or atopic dermatitis.
A method for producing an antibody, comprising culturing the cell of claim 9 under appropriate conditions for the production of the antibody, and isolating the produced antibody.

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