RU2816207C2 - Anti-ige antibodies - Google Patents

Abstract

FIELD: biotechnology; chemistry.

SUBSTANCE: invention relates to biochemistry, particularly to an anti-IgE antibody or an antigen-binding agent. Also disclosed is an isolated DNA molecule coding said antibody; a vector for cloning or expressing said antibody; pharmaceutical composition containing said antibody. Disclosed is the use of said antibodies and compositions for treatment.

EFFECT: invention enables effective treatment of diseases using said anti-IgE antibody.

12 cl, 16 dwg, 13 tbl, 8 ex

Description

Field of technology to which the invention relates

The present invention relates to the field of improved anti-IgE antibodies and antigen binding fragments, as well as compositions thereof, that target IgE, for example, for use in the treatment of diseases caused by IgE (such as allergic reactions or certain autoimmune reactions), and in particular , diseases caused by the interaction of IgE and the FcεRI receptor. In particular, the present invention relates to improved anti-IgE antibodies and antigen binding fragments related to new mutants of omalizumab ( ^Xolair® ). The improved anti-IgE antibodies and antigen-binding fragments of the invention may have improved affinity for IgE and/or improved interaction with the Cε2 domain of IgE, and/or an improved modified epitope on IgE (for example, further including the Cε2 domain of IgE), and/or the ability cause dissociation of IgE from the FcεRI receptor at pharmaceutically usable concentrations. In one aspect, improved or new methods of treating IgE-mediated diseases in which IgE is the target (eg, free IgE and/or IgE complexed with the FcεRI receptor) are disclosed.

Prerequisites for creating the invention

IgE is a member of the immunoglobulin family that mediates allergic reactions such as asthma, food allergies, type 1 hypersensitivity, and familial sinusitis, which are ubiquitous. IgE is secreted by B cells and expressed on their surface. IgE synthesized by B cells is anchored to the B cell membrane by a transmembrane domain linked to the mature IgE sequence by a short membrane-bound region. IgE also associates with B cells (as well as monocytes, eosinophils, and platelets) through binding of its Fc region to the low-affinity IgE receptor (FcεRII). When a mammal is exposed to an allergen, B cells proliferate clonally and produce IgE, which binds the allergen. This IgE is in turn released into the circulation by B cells, where it binds to B cells (via FcεRII) as well as mast cells and basophils through the so-called high-affinity receptor (FcεRI) present on the surface of mast cells and basophils. As a result, mast cells and basophils are sensitized to the allergen. Upon subsequent exposure to the allergen, FcεRI cross-links on these cells and, as a result, activates their release of histamine and other factors that cause clinical hypersensitivity and anaphylaxis.

Omalizumab ( ^Xolair® ) is a recombinant DNA humanized IgG1κ monoclonal antibody that selectively binds to human immunoglobulin E (IgE) [Cε3 domain]. The antibody has a molecular weight of approximately 149 kDa. Xolair ^® is produced in a suspension culture of Chinese hamster ovary cells in a nutrient medium containing the antibiotic gentamicin. Xolair ^® is a sterile, white, preservative-free, lyophilized powder contained in a single-use vial that is reconstituted with Sterile Water for Injection (SWI), USP, (or, alternatively, as a liquid preparation in a sterile syringe) and administered subcutaneously (s.c.). j) by injection [see EP602126 (and SPC/GB06/005 based on it); WO93/04173; US6267958 (and Xolair ^® PTE based on this patent); WO97/04807; WO97/04801; Presta et al. (1993) J. Immunol. 151:2623-2632].

Omalizumab is currently indicated for the treatment of moderate to severe chronic asthma in patients who have a positive skin test or in vitro reaction to year-round aeroallergens and who have symptoms that are not adequately controlled with inhaled corticosteroids (from the prescribing information for ^Xolair® ).

Problems associated with the use of omalizumab are: 1) it targets free IgE but does not target (or ineffectively targets) pathogenic variants of the IgE/FcεRI complex at pharmaceutically used doses; 2) perhaps due to the fact that pathogenic variants of the IgE/FcεRI complex are not targeted, “it takes at least 12-16 weeks for the effect of Xolair treatment to appear” (Xolair ^® 150 mg solution - Brief characteristics of the drug, 2014) -or, in fact, to determine whether Xolair ^® will be effective for a particular patient, or whether other treatment will be necessary; 3) it should not be used in patients with high levels of IgE (eg, because pathogenic variants of the IgE/FcεRI complex are not targeted and do not disappear over time due to high levels of free IgE in the patient); 4) when taking omalizumab, local type I or systemic reactions may occur, including anaphylaxis and anaphylactic shock" (Xolair ^® 150 mg solution - Brief characteristics of the drug, 2014); 5) its affinity for IgE is not very high (about 2 nM).

The purpose of the present invention is to identify new antibodies that solve one or more of these problems.

The next goal is to identify antibodies against new epitopes (that have a stronger interaction with IgE Cε2 than omalizumab) and/or antibodies based on new mutants of omalizumab with increased affinity and/or increased ability to cause dissociation of the IgE/FcεRI complex.

A further object of the invention is to identify new compounds, methods and compositions for the treatment of diseases associated with IgE, in particular diseases associated with the IgE/FcεRI complex, for example, allergic diseases.

SUMMARY OF THE INVENTION

In one aspect, the invention provides an anti-IgE antibody, or antigen binding fragment, that contacts an epitope containing residues T373, W374, S375, R376, A377, S378, G379, P381, Q417, C418, R419, T421, P426, R427, A428 of the Cε3 domain and residues D278 and T281 of the Cε2 domain of human IgE. In further embodiments, the epitope may further comprise one or more of residues K380 and/or M430 of the human IgE Cε3 domain and/or one or more of residues D276, V277, L279, S280, A282 and/or T298 of the human IgE Cε2 domain.

The invention is based on the results of the crystal structure study in Example 1, which for the first time demonstrated the interaction of an improved antibody (omalizumab-based) with IgE-Fc, where significant interactions were observed with the Cε2 domain of IgE in the mutation region. This may result in improved functional characteristics of the anti-IgE antibody, or antigen binding fragment, compared to omalizumab and/or omalizumab Fab. For example, an anti-IgE antibody, or antigen binding fragment, may be capable of causing dissociation of human IgE from FcεRI at concentrations (or peak serum concentrations) of less than 7, 3, 1, 0.66, 0.5 or 0.3 μM ( for example, when determined by the method described in Example 2). For example, an anti-IgE antibody, or antigen binding fragment, may have improved/stronger affinity (lower K _D ) for human IgE (eg, using IgE-Fc) (eg, when determined by the method described in Example 6) compared with omalizumab and/or omalizumab Fab; and/or improved ability to cause dissociation of the IgE/FcεRI complex (eg, when determined by the method described in Example 2) compared to omalizumab and/or omalizumab Fab; and/or the ability to cause dissociation of human IgE from FcεRI at concentrations (or peak serum concentrations) lower than the concentrations of omalizumab and/or omalizumab Fab (eg, as determined by the method described in Example 2). “Improved K _D ” means at least 5, 10, 20, 30, 40 or 50% lower value than the value for omalizumab and/or omalizumab Fab. The K _D of the anti-IgE antibody, or antigen binding fragment, of the invention may be less than 2, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4 or 0.3 nM. “Improved ability or ability” to cause dissociation of human IgE from FcεRI means at least 5, 10, 20, 30, 40, 50, or 100% improved ability compared to omalizumab and/or omalizumab Fab (e.g., as measured by % dissociation and/or the apparent rate of dissociation of the IgE/FcεRI complex, as described in Examples 2 and 7), and/or achieving dissociation at a concentration at which omalizumab and/or omalizumab Fab are unable to cause dissociation.

For the avoidance of doubt, the anti-IgE antibody, or antigen binding fragment, of the invention is not omalizumab or an omalizumab Fab.

In one embodiment, the epitope is determined by crystallography (e.g., as described in Example 1) by identifying IgE residues within 4 or 5 Å of the anti-IgE antibody, or antigen-binding moiety, in the crystal structure of the IgE-Fc/anti-antibody complex. IgE antibody, or antigen-binding fragment. The IgE-Fc region used may have the sequence SEQ ID NO:108 (with additional mutations N265Q and N371Q).

In one embodiment, the anti-IgE antibody, or antigen binding fragment, contacts an epitope at a particular binding site, wherein the Cε3 domain and Cε2 domain portions of the epitope are on different chains of human IgE. IgE has two chains in the Fc domain, each of which has a Cε3 domain and a Cε2 domain.

In one embodiment, the anti-IgE antibody, or antigen binding fragment, contacts an epitope at a particular binding site, wherein the Cε3 domain and Cε2 domain portions of the epitope are on the same human IgE chain.

For the avoidance of doubt, two anti-IgE antibodies, or antigen binding fragments, of the invention can bind to human IgE, but only one of them is required to interact with an epitope of the invention comprising the Cε3 and Cε2 domains (the other can only interact with the other Cε3 domain , For example).

In one embodiment (optionally also including features of the first aspect of the invention), the anti-IgE antibody, or antigen binding fragment, is specific for the specified epitope containing residues T373, W374, S375, R376, A377, S378, G379, P381, Q417 , C418, R419, T421, P426, R427, A428 of the Cε3 domain and residues D278 and T281 of the human IgE Cε2 domain. Optionally, said epitope may further comprise one or more of residues K380 and/or M430 of the human IgE Cε3 domain and/or one or more of residues D276, V277, L279, S280, A282 and/or T298 of the human IgE Cε2 domain. For the avoidance of doubt, an anti-IgE antibody, or antigen-binding fragment, is specific for a specified epitope if it recognizes and binds to a specific human IgE structure containing the specified epitope, and not in general to human IgE.

In a further aspect (optionally also including features of the first aspect of the invention) there is provided an anti-IgE antibody, or antigen binding fragment, comprising a heavy chain variable region containing a complementarity determining region, CDR-H3, with the amino acid sequence of SEQ ID NO:18, and a variable a light chain region containing a complementarity determining region, CDR-L1, with the amino acid sequence of SEQ ID NO:29, wherein the light chain variable region also contains a framework region, FR-L3, with an amino acid sequence selected from SEQ ID NO:32, which has one, two, three, four, five, six, seven or more amino acid substitutions to enhance the interaction of the anti-IgE antibody, or antigen binding fragment, with the Cε2 domain of human IgE.

In a further aspect (optionally also including features of the preceding aspects of the invention) there is provided an anti-IgE antibody, or antigen binding fragment, comprising a heavy chain variable region containing a complementarity determining region, CDR-H3, with the amino acid sequence of SEQ ID NO:18, and a variable a light chain region containing a complementarity determining region, CDR-L1, with the amino acid sequence of SEQ ID NO:29, wherein the light chain variable region also contains a framework region, FR-L1, with the amino acid sequence of SEQ ID NO:28, which has one, two, three, four, five, six, seven or more amino acid substitutions to enhance the interaction of the anti-IgE antibody, or antigen binding fragment, with the Cε2 domain of human IgE.

If the CDR-H3 and CDR-L1 regions anchor and target an anti-IgE antibody, or antigen-binding fragment, to the Cε3 region of the IgE (as in the case of omalizumab), the change(s) in the FR-L3 and/or FR-L1 sequences allows stronger interaction with the Cε2 domain of human IgE. The stronger interaction of a mutant compared to omalizumab or omalizumab Fab can be assessed by measuring the affinity [lower K _D ] (e.g., as determined by the method described in Example 6) and/or the improved dissociation characteristics of the IgE/FcεRI complex (e.g., as determined by the method , described in Example 2).

A stronger interaction of the anti-IgE antibody, or antigen binding fragment, with the Cε2 domain of human IgE can be characterized based on the improved functional characteristics of the anti-IgE antibody, or antigen binding fragment, in comparison with omalizumab and/or omalizumab Fab. For example, an anti-IgE antibody, or antigen binding fragment, may have the ability to cause dissociation of human IgE from FcεRI at concentrations (or peak serum concentrations) of less than 7, 3, 1, 0.66, 0.5 or 0.3 μM ( for example, when determined by the method described in Example 2). For example, an anti-IgE antibody, or antigen binding fragment, may have improved/stronger affinity (lower K _D ) for human IgE (eg, using IgE-Fc) (eg, when determined by the method described in Example 6) compared with omalizumab and/or omalizumab Fab; and/or improved ability to cause dissociation of the IgE/FcεRI complex (eg, when determined by the method described in Example 2) compared to omalizumab and/or omalizumab Fab; and/or the ability to cause dissociation of human IgE from FcεRI at concentrations (or peak serum concentrations) lower than those of omalizumab and/or omalizumab Fab (eg, as determined by the method described in Example 2). “Improved K _D ” means at least 5, 10, 20, 30, 40 or 50% lower value than the value for omalizumab and/or omalizumab Fab. The K _D of the anti-IgE antibody, or antigen binding fragment, of the invention may be less than 2, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4 or 0.3 nM. “Improved ability or ability” to cause dissociation of human IgE from FcεRI means at least 5, 10, 20, 30, 40, 50, or 100% improved ability compared to omalizumab and/or omalizumab Fab (e.g., as measured by % dissociation and/or the apparent rate of dissociation of the IgE/FcεRI complex, as described in Examples 2 and 7), and/or achieving dissociation at a concentration at which omalizumab and/or omalizumab Fab are unable to cause dissociation.

For the avoidance of doubt, the anti-IgE antibody, or antigen binding fragment, of the invention is not omalizumab or an omalizumab Fab.

In one embodiment, the FR-L3 region is mutated from SEQ ID NO:129 at one or more of positions S60, S63, S76, S77 and/or Q79 (Kabat) to one of the other natural amino acids.

For example, the FR-L3 region may have a mutation at position S60 (Kabat) to one of the other naturally occurring amino acids, such as M, R, K, N, Q or T, particularly M.

For example, the FR-L3 region may have a mutation at position S63 (Kabat) to one of the other naturally occurring amino acids, such as W or Y, particularly Y.

For example, the FR-L3 region may have a mutation at position S76 (Kabat) to one of the other naturally occurring amino acids, particularly N.

For example, the FR-L3 region may have a mutation at position S77 (Kabat) to one of the other naturally occurring amino acids, such as R or K, particularly R.

For example, the FR-L3 region may have a mutation at position Q79 (Kabat) to one of the other naturally occurring amino acids, such as R or K, particularly R.

For example, the FR-L1 region may be mutated from SEQ ID NO:20 at position G16 and/or R18 (Kabat) to one of the other natural amino acids.

In specific embodiments, the amino acid sequence of the mutant anti-IgE antibody FR-L3 region, or antigen binding fragment, is selected from SEQ ID NOs: 43-49, 60-83, 131, or 138.

In a further embodiment, the FR-L3 region is also mutated from SEQ ID NO:129 at position S67 (Kabat) to one of the other naturally occurring amino acids to increase its affinity (lower K _D ) for human IgE. In this case, the mutation may result in increased interaction of the anti-IgE antibody, or antigen-binding fragment, with the Cε3 domain of IgE. For example, the FR-L3 region may have a mutation at position S67 (Kabat) to M (especially), E, or D. In particular embodiments, the amino acid sequence of the mutated FR-L3 region of the anti-IgE antibody, or antigen binding fragment, is selected from SEQ ID NO:53-59, 84-107, 131 or 138.

The anti-IgE antibody, or antigen binding fragment, of the invention may have a light chain variable region also comprising a complementarity determining region, CDR-L2, with the amino acid sequence of SEQ ID NO:31.

In one embodiment, the CDR-L2 region is mutated at position S52 (Kabat) to one of the other naturally occurring amino acids to increase its affinity (lower K _D ) for human IgE. In this case, the mutation may result in increased interaction of the anti-IgE antibody, or antigen-binding fragment, with the Cε3 domain of IgE. For example, the CDR-L2 region may have a mutation relative to SEQ ID NO:129 at position S52 (Kabat) to D (especially), E, Q, or R. In particular embodiments, the amino acid sequence of the mutated CDR-L2 region is selected from SEQ ID NO :50 or SEQ ID NO:51.

The anti-IgE antibody, or antigen binding fragment, of the invention may have a heavy chain variable region also comprising a complementarity determining region, CDR-H1, with the amino acid sequence of SEQ ID NO:14.

The anti-IgE antibody, or antigen binding fragment, of the invention may have a heavy chain variable region also comprising a complementarity determining region, CDR-H2, with the amino acid sequence of SEQ ID NO:16.

The anti-IgE antibody, or antigen binding fragment, of the invention may have a light chain variable region also comprising a complementarity determining region, CDR-L3, with the amino acid sequence of SEQ ID NO:33.

The anti-IgE antibody, or antigen binding fragment, of the invention may have a heavy chain variable region also comprising a framework region, FR-H1, with the amino acid sequence of SEQ ID NO:13.

The anti-IgE antibody, or antigen binding fragment, of the invention may have a heavy chain variable region also comprising a framework region, FR-H2, with the amino acid sequence of SEQ ID NO:15.

The anti-IgE antibody, or antigen binding fragment, of the invention may have a heavy chain variable region also comprising a framework region, FR-H3, with the amino acid sequence of SEQ ID NO:17.

The anti-IgE antibody, or antigen binding fragment, of the invention may have a heavy chain variable region also comprising a framework region, FR-H4, with the amino acid sequence of SEQ ID NO:19.

The anti-IgE antibody, or antigen binding fragment, of the invention may have a light chain variable region also comprising a framework region, FR-L2, with the amino acid sequence of SEQ ID NO:30.

The anti-IgE antibody, or antigen binding fragment, of the invention may have a light chain variable region also comprising a framework region, FR-L4, with the amino acid sequence of SEQ ID NO:34.

The anti-IgE antibody, or antigen binding fragment, of the invention may have a light chain variable region, VL, having an amino acid sequence selected from SEQ ID NO:35, SEQ ID NO:132 or SEQ ID NO:134, or SEQ ID NO:141 , or SEQ ID NO:144, or SEQ ID NO:145, or SEQ ID NO:158, or SEQ ID NO:159.

The anti-IgE antibody, or antigen binding fragment, of the invention may have a heavy chain variable region, VH, having the amino acid sequence of SEQ ID NO:1.

The anti-IgE antibody, or antigen binding fragment, of the invention may further comprise a light chain constant region.

The anti-IgE antibody, or antigen binding fragment, of the invention may have a light chain constant region that is a kappa constant region.

The anti-IgE antibody, or antigen binding fragment, of the invention may have a light chain constant region having the L154P mutation (Kabat).

The anti-IgE antibody, or antigen binding fragment, of the invention may have a light chain variable region and a light chain constant region, VL-CL, having an amino acid sequence selected from SEQ ID NO:39 or SEQ ID NO:41, or SEQ ID NO: 117, or SEQ ID NO:119, or SEQ ID NO:125, or SEQ ID NO:127, or SEQ ID NO:136, or SEQ ID NO:143, optionally containing a signal sequence having the amino acid sequence of SEQ ID NO:160 .

The anti-IgE antibody, or antigen binding fragment, of the invention may further comprise a heavy chain constant region, CH1.

The anti-IgE antibody, or antigen binding fragment, of the invention may have a heavy chain variable region and a heavy chain constant region, VH-CH1, having the amino acid sequence of SEQ ID NO:5.

The anti-IgE antibody, or antigen binding fragment, of the invention may further comprise a heavy chain Fc region, Fc.

The anti-IgE antibody, or antigen binding fragment, of the invention may have an Fc of human IgG1 or human IgG4.

The anti-IgE antibody, or antigen binding fragment, of the invention may have a heavy chain variable region, a heavy chain constant region, and a heavy chain Fc region, VH-CH1-Fc, having the amino acid sequence of SEQ ID NO:9.

A further aspect of the invention provides an anti-IgE antibody, or antigen binding fragment, comprising a heavy chain variable region containing a complementarity determining region, CDR-H3, with the amino acid sequence of SEQ ID NO:18, and a light chain variable region containing a complementarity determining region, CDR -L1, with the amino acid sequence SEQ ID NO:29, wherein:

a. the light chain variable region also contains a framework region, FR-L3, with the amino acid sequence of SEQ ID NO:32, wherein the FR-L3 region is mutated from SEQ ID NO:129 at position S67 (Kabat) to one of the other naturally occurring amino acids to increase affinity (lower K _D ) anti-IgE antibody, or antigen binding fragment, for human IgE; and/or

b. the light chain variable region also contains a complementarity determining region, CDR-L2, with the amino acid sequence of SEQ ID NO:31, wherein the CDR-L2 region is mutated from SEQ ID NO:129 at position S52 (Kabat) to one of the other natural amino acids to increase affinity (lower K _D ) of an anti-IgE antibody, or antigen-binding fragment, for human IgE.

The present inventors have discovered that either or both of these mutations can remarkably increase the affinity (improved or lower _KD ) of an omalizumab-based anti-IgE antibody or antigen-binding fragment for human IgE (e.g., when using IgE -Fc) (for example, when determined by the method described in Example 6). In particular, there is an increase in affinity compared to omalizumab and/or omalizumab Fab. Mutations may lead to improved interaction with the Cε3 domain of IgE. “Improved or lower K _D ” means at least 5, 10, 20, 30, 40 or 50% lower value than the value for omalizumab and/or omalizumab Fab. The K _D of the anti-IgE antibody, or antigen binding fragment, of the invention may be less than 2, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4 or 0.3 nM.

For example, the FR-L3 region may have a mutation relative to SEQ ID NO:129 at position S67 (Kabat) to M (in particular), E or D.

In specific embodiments, the amino acid sequence of the mutant anti-IgE antibody FR-L3 region, or antigen binding fragment, is selected from SEQ ID NOs: 52-59, 84-107, 131, or 138.

For example, the CDR-L2 region may have a mutation relative to SEQ ID NO:129 at position S52 (Kabat) to D (in particular), E, Q or R.

In specific embodiments, the amino acid sequence of the mutant anti-IgE antibody CDR-L2 region, or antigen binding fragment, is selected from SEQ ID NO:50 (in particular) or SEQ ID NO:51.

The anti-IgE antibody, or antigen binding fragment, may have a heavy chain variable region also comprising a complementarity determining region, CDR-H1, with the amino acid sequence of SEQ ID NO:14.

The anti-IgE antibody, or antigen binding fragment, may have a heavy chain variable region also comprising a complementarity determining region, CDR-H2, with the amino acid sequence of SEQ ID NO:16.

The anti-IgE antibody, or antigen binding fragment, may have a light chain variable region also comprising a complementarity determining region, CDR-L3, with the amino acid sequence of SEQ ID NO:33.

The anti-IgE antibody, or antigen binding fragment, may have a heavy chain variable region also comprising a framework region, FR-H1, with the amino acid sequence of SEQ ID NO:13.

The anti-IgE antibody, or antigen binding fragment, may have a heavy chain variable region also comprising a framework region, FR-H2, with the amino acid sequence of SEQ ID NO:15.

The anti-IgE antibody, or antigen binding fragment, may have a heavy chain variable region also comprising a framework region, FR-H3, with the amino acid sequence of SEQ ID NO:17.

The anti-IgE antibody, or antigen binding fragment, may have a heavy chain variable region also comprising a framework region, FR-H4, with the amino acid sequence of SEQ ID NO:19.

The anti-IgE antibody, or antigen binding fragment, may have a light chain variable region also comprising a framework region, FR-L2, with the amino acid sequence of SEQ ID NO:30.

The anti-IgE antibody, or antigen binding fragment, may have a light chain variable region also comprising a framework region, FR-L4, with the amino acid sequence of SEQ ID NO:34.

The anti-IgE antibody, or antigen binding fragment, may have a light chain variable region, VL, comprising the sequential regions FR-L1, CDR-L1, FR-L2, CDR-L2, FR-L3, CDR-L3 and FR-L4, and having the amino acid sequence of SEQ ID NO:20, except that the CDR-L2 region has an amino acid sequence selected from SEQ ID NO:50 (in particular) or SEQ ID NO:51.

The anti-IgE antibody, or antigen binding fragment, may have a light chain variable region, VL, comprising the sequential regions FR-L1, CDR-L1, FR-L2, CDR-L2, FR-L3, CDR-L3 and FR-L4, and having the amino acid sequence of SEQ ID NO:20, except that the FR-L3 region has the amino acid sequence of SEQ ID NO:52.

The anti-IgE antibody, or antigen binding fragment, may have a light chain variable region, VL, comprising the sequential regions FR-L1, CDR-L1, FR-L2, CDR-L2, FR-L3, CDR-L3 and FR-L4, and having the amino acid sequence of SEQ ID NO:20, except that the CDR-L2 region has an amino acid sequence selected from SEQ ID NO:50 (in particular) or SEQ ID NO:51, and the FR-L3 region has an amino acid sequence that selected from SEQ ID NO:52, 131 or 138.

The anti-IgE antibody, or antigen binding fragment, of the invention may have a heavy chain variable region, VH, having the amino acid sequence of SEQ ID NO:1.

The anti-IgE antibody, or antigen binding fragment, may further comprise a light chain constant region.

The anti-IgE antibody, or antigen binding fragment, may have a light chain constant region that is a kappa constant region.

The anti-IgE antibody, or antigen binding fragment, may have a light chain variable region and a light chain constant region, VL-CL, having the amino acid sequence of SEQ ID NO:24, except that the CDR-L2 region has an amino acid sequence selected from SEQ ID NO:50 (specifically) or SEQ ID NO:51.

The anti-IgE antibody, or antigen binding fragment, may have a light chain variable region and a light chain constant region, VL-CL, having the amino acid sequence of SEQ ID NO:24, except that the FR-L3 region has the amino acid sequence of SEQ ID NO: 52.

The anti-IgE antibody, or antigen binding fragment, may have a light chain variable region and a light chain constant region, VL-CL, having the amino acid sequence of SEQ ID NO:24, except that the CDR-L2 region has an amino acid sequence selected from SEQ ID NO:50 (in particular) or SEQ ID NO:51, and the FR-L3 region has an amino acid sequence selected from SEQ ID NO:52, 131 or 138.

In a further aspect, the invention provides an anti-IgE antibody, or antigen binding fragment, comprising a heavy chain variable region and a light chain variable region, wherein:

a. the heavy chain variable region contains CDR-H1 with the amino acid sequence of SEQ ID NO:14, CDR-H2 with the amino acid sequence of SEQ ID NO:16 and CDR-H3 with the amino acid sequence of SEQ ID NO:18, and the light chain variable region contains CDR-L1 with the amino acid sequence of SEQ ID NO:29, CDR-L2 with the amino acid sequence of SEQ ID NO:50, CDR-L3 with the amino acid sequence of SEQ ID NO:33 and the FW-L3 framework region with the amino acid sequence of SEQ ID NO:131 or 138; or

b. the heavy chain variable region contains the amino acid sequence of SEQ ID NO:1 and the light chain variable region contains the amino acid sequence selected from SEQ ID NO:132 or 139.

In one embodiment, the anti-IgE antibody, or antigen binding fragment, may further comprise a light chain constant region, wherein the light chain variable region and the VL-CL light chain constant region have an amino acid sequence selected from SEQ ID NO: 136 or 143, optionally comprising a signal sequence having the amino acid sequence of SEQ ID NO:160.

In all embodiments described herein, the anti-IgE antibody, or antigen binding fragment, may further comprise a heavy chain constant region, CH1.

The anti-IgE antibody, or antigen binding fragment, may have a heavy chain variable region and a heavy chain constant region, VH-CH1, having the amino acid sequence of SEQ ID NO:5.

The anti-IgE antibody, or antigen binding fragment, may further comprise a heavy chain Fc region, Fc.

The anti-IgE antibody, or antigen binding fragment, may have an Fc of human IgG1 or human IgG4.

The anti-IgE antibody, or antigen binding fragment, may have a heavy chain variable region, a heavy chain constant region, and a heavy chain Fc region, VH-CH1-Fc, having the amino acid sequence of SEQ ID NO:9.

The anti-IgE antibody, or antigen binding fragment, in all aspects of the invention can be selected from the group consisting of: a whole antibody molecule having full length heavy and light chains, or a fragment thereof.

The anti-IgE antibody, or antigen binding fragment, of the invention may be selected from the group consisting of: Fab fragment, modified Fab' fragment, Fab' fragment, F(ab') ₂ fragment, Fv, scFv, scab, diabody , bispecific antibody, tribody, FabFv, Fab-Fv-Fv, tribody or (Fab-Fv) ² -Fc. Without being bound by specific theory, an anti-IgE antibody, or antigen binding fragment, of the invention may be associated with a lower risk of causing anaphylaxis if it has only one rather than multiple IgE antigen binding sites.

In one embodiment, the anti-IgE antibody is a Fab fragment linked directly or via a linker to an scFv fragment that binds to a serum carrier protein, such as human serum albumin.

In one embodiment, the scFv may comprise a heavy chain variable region and a light chain variable region, preferably linked through a linker having SEQ ID NO:151, wherein the heavy chain variable region comprises a CDR-H1 with the amino acid sequence of SEQ ID NO:152, CDR-H2 having the amino acid sequence SEQ ID NO:153 and CDR-H3 having the amino acid sequence SEQ ID NO:154, and the light chain variable region contains CDR-L1 having the amino acid sequence SEQ ID NO:155, CDR-L2 having the amino acid sequence SEQ ID NO:156, CDR-L3 with amino acid sequence SEQ ID NO:157.

In one embodiment, the scFv fragment has the amino acid sequence of SEQ ID NO:150. In one preferred embodiment, the Fab fragment comprises a heavy chain variable region and a light chain variable region, wherein:

a. the heavy chain variable region contains CDR-H1 with the amino acid sequence of SEQ ID NO:14, CDR-H2 with the amino acid sequence of SEQ ID NO:16 and CDR-H3 with the amino acid sequence of SEQ ID NO:18, and the light chain variable region contains CDR-L1 with the amino acid sequence of SEQ ID NO:29, CDR-L2 with the amino acid sequence of SEQ ID NO:50, CDR-L3 with the amino acid sequence of SEQ ID NO:33 and the FW-L3 framework region with the amino acid sequence of SEQ ID NO:131 or 138; or

b. the heavy chain variable region contains the amino acid sequence of SEQ ID NO:1 and the light chain variable region contains the amino acid sequence selected from SEQ ID NO:132 or 139.

In another embodiment, the Fab fragment also comprises a heavy chain constant region and a light chain constant region, wherein the heavy chain variable region and the VL-CH1 heavy chain constant region have the amino acid sequence of SEQ ID NO:5, and wherein the light chain variable region and the VL-CL light chain constant region have an amino acid sequence selected from SEQ ID NO:136 or 143, optionally containing a signal sequence that has the amino acid sequence of SEQ ID NO:160.

In another embodiment, the scFv fragment is linked to the CH1 Fab fragment through a linker having the amino acid sequence of SEQ ID NO:149.

In one embodiment, the heavy chain variable region and the heavy chain constant region, linker and scFv have the amino acid sequence of SEQ ID NO:147, optionally comprising a signal sequence that has the amino acid sequence of SEQ ID NO:160.

In another embodiment, the heavy chain of the Fab fragment associated with the scFv of SEQ ID NO:147 is paired with a light chain variable and constant region that have SEQ ID NO:136 or 143.

The anti-IgE antibody, or antigen binding fragment, of the invention may have an effector or reporter molecule attached thereto.

The anti-IgE antibody, or antigen binding fragment, of the invention may be glycosylated (eg within the Fc domain) and/or may be conjugated to a polymer selected from starch, albumin and polyethylene glycol (PEG). In one embodiment, the conjugated PEG may have a molecular weight in the range of 5-50 kDa.

The anti-IgE antibody, or antigen binding fragment, of the invention may be humanized.

Another aspect of the invention relates to an isolated DNA sequence encoding the heavy and/or light chain(s) of an anti-IgE antibody, or antigen binding fragment, of the invention. Also provided is a cloning or expression vector containing one or more DNA sequences of the invention. For example, the cloning or expression vector may contain one or more DNA sequences selected from SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40 or SEQ ID NO:42, or SEQ ID NO:133, or SEQ ID NO:135, or SEQ ID NO:137, or SEQ ID NO:140, or SEQ ID NO:142, or SEQ ID NO:144, and optionally may further comprise one or more DNA sequences selected from SEQ ID NO :2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10 or SEQ ID NO:12, or SEQ ID NO:148.

Another aspect of the invention relates to a host cell containing one or more cloning, or expression, vectors of the invention. The host cell of the invention may optionally also contain one or more cloning, or expression, vectors containing one or more DNA sequences selected from SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10 or SEQ ID NO:12, or SEQ ID NO:148.

Also provided is a method for producing an anti-IgE antibody or antigen binding fragment according to the invention, comprising culturing a host cell according to the invention and isolating an anti-IgE antibody or antigen binding fragment.

The following aspect relates to a pharmaceutical composition containing an anti-IgE antibody, or antigen binding fragment, according to the invention, in combination with one or more of a pharmaceutically acceptable excipient, diluent or carrier. Preferably, the anti-IgE antibody, or antigen binding fragment, of the invention is present at a dose of 50-200, preferably about or exactly 150 mg per ml of diluent. In specific embodiments, the excipient includes one or both of L-arginine, L-histidine. The excipient may, alone or in combination, include polysorbate 20. The diluent may be water or an aqueous isotonic solution.

The pharmaceutical composition of the invention may be presented in a sterile vial as a powder for reconstitution prior to subcutaneous administration, or in a sterile syringe for immediate subcutaneous administration.

The pharmaceutical composition of the invention may contain a total dose of anti-IgE antibody, or antigen binding fragment, of 75-600 mg - for example, about or exactly 100 or 150 mg.

In addition, the pharmaceutical composition of the invention may contain other active ingredients, either together with the anti-IgE antibody or antigen binding fragment, or in a separate preparation for co-administration with the anti-IgE antibody or antigen binding fragment. For example, the pharmaceutical composition of the invention may be used in the context of a specific allergy-based immunotherapy in which an anti-IgE antibody, or antigen binding fragment, of the invention is co-administered with a separate (but possibly co-packaged) allergen. Thus, the pharmaceutical composition of the invention may be intended for specific allergy-based immunotherapy, in which the patient receives the pharmaceutical composition of the invention 7, 6, 5, 4, 3, 2 or 1 days before (or on the same day) administration of the therapeutic allergen.

The anti-IgE antibody or antigen binding fragment or composition of the invention can be used as a medicine.

The anti-IgE antibody, or antigen binding fragment, or composition of the invention can be used for the treatment or prevention of a disease.

The anti-IgE antibody, or antigen binding fragment, or composition of the invention can be used for the treatment or prevention of diseases associated with the human IgE-FcεRI complex.

The anti-IgE antibody or antigen binding fragment or composition of the invention can be used for the treatment or prevention of diseases by promoting the dissociation of the human IgE-FcεRI complex and the binding of human IgE by the anti-IgE antibody or antigen binding fragment.

The anti-IgE antibody, or antigen binding fragment, or composition of the invention can be used for the treatment or prevention of one or more of: allergies; allergic asthma; severe asthma; moderate asthma; chronic spontaneous urticaria; chronic idiopathic urticaria; prolonged allergic rhinitis; seasonal allergic rhinitis; exacerbations of asthma; acute bronchospasm; status asthmaticus; hyper-IgE syndrome; allergic bronchopulmonary aspergillosis; prevention of anaphylactic reactions; food allergies; atopic dermatitis; allergic rhinitis; allergies to bee venom; idiopathic anaphylaxis; peanut allergies; allergies to latex; inflammatory skin diseases; urticaria (caused by sunlight, cold, localized heat and/or prolonged pressure); cutaneous mastocytosis; systemic mastocytosis; eosinophil-related gastrointestinal disease; bullous pemphigoid; interstitial cystitis; nasal polyps; idiopathic angioedema or non-allergic asthma.

Also provided is a method of treating or preventing a disease in a human subject, comprising administering to the subject an effective amount of an anti-IgE antibody, or antigen binding fragment, or composition of the invention. The method may be intended for the treatment or prevention of diseases associated with the human IgE-FcεRI complex. The method of the invention allows for the treatment or prevention of a disease by dissociating the human IgE-FcεRI complex and binding of the human IgE with an anti-IgE antibody, or antigen binding fragment, of the invention.

The method of the invention may be intended for the treatment or prevention of one or more of: allergies; allergic asthma; severe asthma; moderate asthma; chronic spontaneous urticaria; chronic idiopathic urticaria; prolonged allergic rhinitis; seasonal allergic rhinitis; exacerbations of asthma; acute bronchospasm; status asthmaticus; hyper-IgE syndrome; allergic bronchopulmonary aspergillosis; prevention of anaphylactic reactions; food allergies; atopic dermatitis; allergic rhinitis; allergies to bee venom; idiopathic anaphylaxis; peanut allergies; allergies to latex; inflammatory skin diseases; urticaria (caused by sunlight, cold, localized heat and/or prolonged pressure); cutaneous mastocytosis; systemic mastocytosis; eosinophil-related gastrointestinal disease; bullous pemphigoid; interstitial cystitis; nasal polyps; idiopathic angioedema or non-allergic asthma.

The present invention has found that an antibody, or antigen binding fragment, against a first polypeptide that mediates its physiological response by binding to a second polypeptide (such as a receptor) can bind to both the free and bound first polypeptide, stabilizing the conformation such a first polypeptide. Such a stabilized conformation has a weaker binding affinity for the second polypeptide than in the absence of the antibody or antigen binding fragment, thereby triggering more rapid dissociation of the first polypeptide from the second polypeptide.

In this regard, the invention in a further aspect relates to an antibody, or antigen binding fragment, capable of binding free and FcεRI-bound human IgE and stabilizing the conformation of IgE. When IgE is in this conformation, it has weaker binding affinity for FcεRI than in the absence of the antibody or antigen-binding fragment, and FcεRI-bound human IgE dissociates from FcεRI. Optionally, when IgE is in such a conformation, the IgE has a lower binding affinity for omalizumab or a fragment thereof than for an antibody or antigen binding fragment of the invention. For example, the antibody, or antigen binding fragment, is an antibody described herein.

In a further aspect, the invention relates to a method for selecting such antibodies, or antigen binding fragments, as described herein. The method includes:

a. contacting the test antibody, or antigen binding fragment, with a sample containing human IgE linked to human FcεRI;

b. measuring the dissociation constant of the test antibody, or antigen binding fragment, to dissociate human IgE from human FcεRI;

c. comparing the dissociation constant measured in step b) with the dissociation constant of omalizumab, or a fragment thereof, for the dissociation of human IgE from human FcεRI;

d. selecting an antibody or antigen binding fragment if said antibody or antigen binding fragment dissociates IgE from FcεRI faster than omalizumab or a fragment thereof.

Alternatively, a method for selecting antibodies, or antigen binding fragments, of the invention includes:

a. contacting the test antibody, or antigen binding fragment, with a sample containing human IgE linked to human FcεRI;

b. measuring the binding affinity of a test antibody, or antigen binding fragment, for human IgE bound to human FcεRI;

c. comparing the binding affinity measured in step b) with the binding affinity of human IgE for FcεRI;

d. selecting an antibody or antigen binding fragment if said antibody or antigen binding fragment has a higher binding affinity for IgE than the affinity of IgE for FcεRI.

Optionally, the selected antibodies, or antigen-binding fragments, cause IgE still bound to FcεRI to transition into a specific conformation, wherein IgE in said stabilized conformation may dissociate from FcεRI faster than IgE bound to FcεRI in the presence of omalizumab or a fragment thereof; and/or may have a higher binding affinity for the antibody, or antigen binding fragment, than for FcεRI.

In a final aspect, the present invention relates to specific antibodies, or antigen binding fragments, containing:

a. a heavy chain variable region comprising SEQ ID NO:1, and a light chain variable region comprising:

i. SEQ ID NO:109; or

ii. SEQ ID NO:113; or

iii. SEQ ID NO:121; or

iv. SEQ ID NO::132; or

v. SEQ ID NO::139; or

b. SEQ ID NO:5 and

i. SEQ ID NO:24 in which S77 and S79 are replaced by Q;

ii. SEQ ID NO:117 or

iii. SEQ ID NO:125; or

iv. SEQ ID NO::136; or

v. SEQ ID NO:143.

In one embodiment of this latter aspect of the invention, the anti-IgE antibody, or antigen binding fragment, contacts, or contacts and is specific for, an epitope containing, with reference to SEQ ID NO:108, residues T373, W374, S375, R376, A377, S378, G379, P381, Q417, C418, R419, T421, P426, R427, A428 of the Cε3 domain and residues D278 and T281 of the human IgE Cε2 domain.

BRIEF DESCRIPTION OF THE DRAWINGS

References and SEQ IDs are provided in the Examples with reference to the Figures.

Figure 1. Fab3 of omalizumab contains three point mutations. Omalizumab Fab3 is derived from omalizumab and contains three point mutations distal to the antigen-binding CDR regions, two in the framework region of the V _L domain (Ser81Arg, Gln83Arg) and one in the Cκ domain (Leu158Pro). The heavy and light chains are marked in white and blue, respectively. Mutant residues are marked in red, and CRDL1 is marked in green to indicate Fab orientation.

Figure 2. General structure of IgE-Fc in complex with Fab3 of omalizumab. (A) Fab3 of omalizumab binds to IgE-Fc in a 2:1 stoichiometry. Fab ¹ (green) binds to IgE-Fc chain B (pink) exclusively through the Cε3 domain. Fab ² (blue) interacts with IgE-Fc chain A (yellow) through the Cε3 domain and interacts slightly with the Cε2 domain from IgE-Fc chain B (pink). (B) The two Fabs form a pseudosymmetric complex approximately twice the size of the Fcε3-4 region axis. For clarity, Cε2 domains are not shown. (C) IgE-Fc is asymmetrically curved in complex with Fab3 of omalizumab. The Cε2 domain from chain B (pink) contacts Fab ² (blue).

Figure 3. Contact surface between Fab3 of omalizumab and IgE-Fc. Shown is the interface between Fab ² Fab3 of omalizumab (heavy and light chains in green and yellow, respectively) and the Cε3 domain of IgE-Fc (pink). Residue labeling for omalizumab Fab3 and Cε3 domain is shown in blue and black, respectively. Hydrogen bonds and van der Waals forces act on the contact surface. A notable feature of the contact interface is the cation-π interaction between Arg419 (Cε3 domain) and Phe103 (CDRH3 Fab3 of omalizumab). The side chain of Phe103 is mainly embedded in a pocket created by Thr373, Trp374, Ser375, Gln417 and Arg419 (Cε3 domain).

Figure 4. Omalizumab Fab3 and DARPin E2_79 bind to overlapping contact surfaces. Both Fab3 of omalizumab and DARPin E2_79 ²⁰ bind to the Cε3 domain. IgE-Fc residues that form only part of the contact surface with Fab3 of omalizumab are marked in orange, while those that form only part of the contact surface with DARPin E2_79, which includes part of the Cε3-Cε4 linker, are marked in blue. The IgE-Fc residues marked in pink, which include Arg419 and Met430, are common to the interfaces with Fab3 of omalizumab and DARPin E2_79.

Figure 5. Conformational flexibility in IgE-Fc. (A) Lateral view of free IgE-Fc ⁸ showing its sharp, asymmetric bend. (B) Frontal view of free IgE-Fc (image rotated 90° counterclockwise from the image in (A). (C) Lateral view of IgE-Fc complexed with Fab3 of omalizumab, showing its partially curved conformation. (D) Frontal view IgE-Fc complexed with Fab3 of omalizumab (image rotated 90° counterclockwise from the image in (C). (E) Lateral view of fully extended IgE-Fc captured by anti-IgE-Fc Fab (Fab3 of omalizumab) ^16. (F ) Frontal view of extended IgE-Fc (image rotated 90° counterclockwise from image in (E).

Figure 6. Conformational flexibility in IgE-Fc. IgE‐Fc flexibility and extension from a bent to a fully extended conformation has previously been studied by molecular dynamics ¹⁶ . IgE-Fc extension is reported as surface free energy as previously described ¹⁶ . (A) Extended conformation of IgE-Fc trapped in the crystal structure of the omalizumab/IgE-Fc ¹⁶ Fab3 complex. (B) Partially curved conformation of IgE-Fc observed in the crystal structure of the Fab3 omalizumab/IgE-Fc complex. (C) Bent conformation of free IgE-Fc ^7,8 . The bent conformation of IgE-Fc occupies the lowest energy level, while the partially bent conformation observed in the Fab3 omalizumab/IgE-Fc complex occupies a clearly different energy level (B).

Figure 7. Disruption of the interaction between IgE-Fc and FcεRI. When complexed with Fab3 of omalizumab, the Cε3 domains adopt the most open conformation known to date for IgE-Fc, which prevents binding to FcεRIα. The structure of IgE-Fc in complex with FcεRIα ⁸ is indicated in yellow, and the two receptor binding subsites are indicated. The structure of Fab3 of omalizumab in complex with IgE-Fc was superimposed on the Cε4 domains, and the Cε3 domains are indicated in blue. The positions of His424 and Pro426 in the two structures are indicated to highlight the different positions adopted by the Cε3 domains.

Figure 8. Disruption of the interaction between IgE-Fc and CD23. (A) Residues of the Cε3 domain that are common to the contact surfaces of both omalizumab Fab3 and CD23 are indicated in pink. (B) Composite images of Cε3 domains (dark gray) from the omalizumab/IgE-Fc Fab3 complex and the CD23/Fcε3-4 complex ¹¹ revealed competition between CD23 (yellow) and omalizumab Fab3 (pink).

Figure 9. Omalizumab Fab3 interaction study with IgE-Fc. (A) Binding of omalizumab Fab3 to IgE-Fc captured via the C-terminal His tag; Omalizumab Fab3 was passed over IgE-Fc at the following concentrations: 100 nM (black), 50 nM (red), 25 nM (green), 12.5 nM (blue), 6.2 nM (cyan), 3.1 nM (purple), 1.6 nM (crimson) and 0.8 nM (dark red). Standard double reference methods were used ³⁶ ; experiments for each concentration were performed in duplicate. (B) Binding of the second Fab3 binding site of omalizumab was characterized using SPR in a sandwich binding format. IgE-Fc was captured on the surface of omalizumab Fab3, and then a second omalizumab Fab3 molecule was added to the omalizumab IgE-Fc/Fab3 complex at concentrations of 1000 nM (black), 500 nM (red), 250 nM (green), 125 nM (blue) , 62.5 nM (cyan), 31.2 nM (violet), 15.6 nM (raspberry), 7.8 nM (dark red), and 0 nM (dark blue). (C) Comparison of the ability of omalizumab Fab3 to bind IgE-Fc captured via the C-terminal His tag (red) and IgE-Fc captured via binding to FcεRIα (blue); in each case, a series of twofold dilutions was tested, with the highest concentration being 1000 nM. The inset shows that Fab3 of omalizumab can still bind to the IgE-Fc/FcεRIα complex, but with a low _Bmax value. (D) Accelerated dissociation of the IgE-Fc/FcεRIα complex mediated by Fab3 of omalizumab at increasing concentrations. First, a 1:1 IgE-Fc/FcεRIα complex was formed by trapping IgE-Fc on immobilized FcεRIα, followed by Fab3 binding of omalizumab at the following concentrations: 5000 nM (magenta), 1000 nM (purple), 200 nM (cyan), 40 nM (blue), 8 nM (green), 1.6 nM (red), and 0 nM (black). The inset shows an enlarged graph of the accelerated dissociation process. Experiments for all concentrations were carried out in duplicate. All binding experiments were performed at 25°C, except those characterizing the second Fab3 binding site of omalizumab (Figure 4B), which were performed at 5°C to minimize allosteric interaction between the two sites.

Figure 10. Direct binding assay, competition experiments, and accelerated dissociation. Direct IgE-Fc binding to immobilized omalizumab Fab3 (A), omalizumab Fab (B), and intact omalizumab (C) was measured. Fab fragments or intact antibody were covalently immobilized at low density using an amino linkage kit (GE Healthcare); IgE-Fc was passed over surfaces at various concentrations using serial 2-fold dilutions with a highest concentration of 100 nM. Experiments for all concentrations were carried out in duplicate. (D) TR-FRET experiments were performed to competitively bind IgE-Fc between omalizumab Fab3 and the αγ fusion protein. Binding between terbium-labeled αγ-fusion protein and Alexa Fluor 647-labeled IgE-Fc was measured at increasing concentrations of unlabeled Fab3 omalizumab inhibitor: 0 μM (black), 2.5 nM (blue), 5 nM (green), 10 nM ( crimson), 20 nM (red). As an inhibitor, omalizumab Fab affects both the apparent _KD and _Bmax of the interaction between IgE-Fc and αγ-fusion protein, suggesting some allosteric inhibition properties. (E) Comparison of accelerated dissociation of the IgE-Fc/sFcεRIα complex mediated by intact omalizumab (black), omalizumab Fab (red), or omalizumab Fab3 (blue), each at a concentration of 5 μM.

Figure 11. Representative electron density map. A stereo image of a 2F _o -F _c electron density map drawn at a trim level of 1.1σ is shown for part of the Cε3 domain of chain A and the covalently N-linked oligosaccharide moiety on Asn394.

Figure 12. Sensogram of IgE-Fc dissociation from immobilized sFcεRIεα obtained using Biacore. Dissociation was monitored in the presence of running buffer (solid line) or IgE binding partners (all other sensorgrams). The analysis was carried out as described in the analysis method (1) of Example 2.

Figure 13. Sensogram of IgE-Fc dissociation from immobilized sFcεRIα obtained using Biacore. Dissociation was monitored in the presence of control supernatant (solid line) or IgE binding partners (all other sensorgrams). The analysis was carried out as described in the analysis method (2) of Example 2.

Figure 14. Dissociation assay of Alexa 488-labeled IgE-Fc from the surface of RBL-SX38 cells. The measured binding data were normalized to 100% at t=0 and the dissociation data were plotted as the change in the proportion of IgE-Fc remaining bound as a function of time.

Figure 15. Analysis of the effect of therapeutic dosing of wild-type Fab omalizumab and Fab3 omalizumab in a 72-hour PCA model.

Figure 16. Residues 224-547 of the wild-type human IgE-Fc sequence (shown in SEQ ID NO:108), with residues 224 and 547 in bold. Numbering follows Dorrington and Bennich (1978) Immunol. Rev. 41:3-25, whereby the L (Leu; leucine) after L253 is designated L235a (boxed) and the next residue is C254. The remaining residues are numbered sequentially without additional additions. Epitope residues are marked with an asterisk (*).

DETAILED DESCRIPTION OF THE INVENTION

The antibody amino acid numbering will be used herein from either the continuous amino acid sequence of an antibody (e.g., omalizumab containing the VH sequence of SEQ ID NO:1 and the VL sequence of SEQ ID NO:20 or SEQ ID NO:129) - so-called "pdb" numbering - or the generally accepted Kabat numbering system may be used. In the case of describing the conventional parts (CDR - complementarity determining regions or FR - framework regions) of the VH or VL sequence of an immunoglobulin, they are linked in a standard order (VH = FR-H1.CDR-H1.FR-H2.CDR-H2.FR-H3 .CDR-H3.FR-H4;VL=FR-L1.CDR-L1.FR-L2.CDR-L2.FR-L3.CDR-L3.FR-L4). For omalizumab, the numbering of the “pdb” portions of the VH (SEQ ID NO:1) is as follows: FR-H1 (amino acids 1-25), CDR-H1 (26-36), FR-H2 (37-50), CDR-H2 ( 51-66), FR-H3 (67-98), CDR-H3 (99-110), FR-H4 (111-121); in this case, the Kabat numbering is as follows: FR-H1 (amino acids 1-25), CDR-H1 (26-35), FR-H2 (36-49), CDR-H2 (50-65), FR-H3 (66- 94), CDR-H3 (95-102), FR-H4 (103-113). For omalizumab, the numbering of the “pdb” portions of the VL (SEQ ID NO:20) is as follows: FR-L1 (amino acids 1-23), CDR-L1 (24-38), FR-L2 (39-53), CDR-L2 ( 54-60), FR-L3 (61-92), CDR-L3 (93-101), FR-L4 (102-111); in this case, the Kabat numbering is as follows: FR-L1 (amino acids 1-23), CDR-L1 (24-34), FR-L2 (35-49), CDR-L2 (50-56), FR-L3 (57- 88), CDR-L3 (89-97), FR-L4 (98-107).

The numbering for IgE antibodies is as described in Dorrington & Bennich (1978) Immunol. Rev. 41:3-25. Thus, the IgE-Fc polypeptides used in this invention (see SEQ ID NO:108) consist of V224-K547 (including the C225A mutation). As shown in Figure 16, the numbering used corresponds to the numbering in Dorrington and Bennich (1978) Immunol. Rev. 41:3-25, where L (Leu, leucine) after position 253 is designated L253a and the remaining residues are numbered sequentially after L253a as C254 and so on. In crystallographic experiments, the following mutations were also introduced into IgE-Fc to simplify the glycosylation pattern: N265Q and N371Q. It is generally accepted that the Cε2 region of IgE-Fc occupies the sequence S226-D330. As used herein, a reference to IgE may be a reference to human IgE (and vice versa), and may also be a reference to IgE-Fc in the context of the assays and methods described herein. The sequence of the Fab arms of full-length human IgE antibodies is not included in this description because they are not present in the crystal structures.

As used herein, “omalizumab” means the commercially available drug ^Xolair® or a full-length IgG antibody comprising a heavy chain containing the amino acid sequence VH SEQ ID NO:1 and a light chain containing the amino acid sequence VL SEQ ID NO:20; or a full-length IgG antibody containing a heavy chain containing the amino acid sequence VH-CH1 SEQ ID NO:5, and a light chain containing the amino acid sequence VL-CL SEQ ID NO:24; or a full-length IgG antibody containing a heavy chain containing the amino acid sequence VH-CH1-Fc SEQ ID NO:9, and a light chain containing the amino acid sequence VL-CL SEQ ID NO:24. "Omalizumab Fab" means a Fab fragment containing a heavy chain containing the amino acid sequence VH SEQ ID NO:1, and a light chain containing the amino acid sequence VL SEQ ID NO:20; or (in particular) a Fab fragment containing a heavy chain containing the amino acid sequence VH-CH1 SEQ ID NO:5, and a light chain containing the amino acid sequence VL-CL SEQ ID NO:24.

General definitions

Unless otherwise indicated, all technical and scientific terms used herein have the meaning commonly given to them by those skilled in the art to which this invention relates. Although methods and materials similar or equivalent to those described herein may be used in practicing or testing the present invention, suitable methods and materials are described herein. All publications, patent applications, patents and other literature references herein are incorporated by reference in their entirety. Furthermore, the materials, methods, and examples are illustrative only and are not intended to be limiting.

Unless the context otherwise requires, terms in the singular shall also include terms in the plural, and terms in the plural shall also include terms in the singular. In this application, the conjunction “or” used means “and/or” unless otherwise indicated. In addition, the term “including” as used, as well as its other forms such as “includes” and “included”, is not limiting. Also, terms such as “element” or “component” cover both elements and components containing a single unit, as well as elements and components containing more than one subunit, unless specifically stated otherwise.

In general, the nomenclatures used, as well as the cell and tissue culture, molecular biology, immunology, microbiology, genetics, protein and nucleic acid chemistry, and hybridization techniques described herein are well known to those skilled in the art and are routinely used in the art. The methods and techniques of the present invention are generally used in a manner generally known in the art and as described in various general and specialized literature cited and discussed throughout this specification unless otherwise indicated. Enzymatic reactions and purification methods can be performed in accordance with the manufacturer's instructions, as is generally accepted in the art or as described herein. The nomenclatures used, as well as the laboratory procedures and methods of analytical chemistry, organic synthesis chemistry, and medicinal and pharmaceutical chemistry described herein, are well known and widely used in the art. Standard techniques are used for chemical synthesis, chemical analyses, formulation, formulation and delivery of pharmaceuticals, and for patient treatment.

Below is an explanation of some terms for a better understanding of the present invention.

As used herein, the term “host” generally means a human subject, and particularly when a human or humanized antibody framework is used as the acceptor structure. Those skilled in the art will appreciate that if a different host is being treated, targeted changes to the antibody, or antigen binding fragment, may be required for that host to avoid rejection or to achieve greater compatibility. Methods are known to use the CDR regions of the present invention and genetically engineer them into an appropriate scaffold or peptide sequence to achieve intended delivery and function in a variety of hosts. Other hosts may include mammals or other vertebrate species. Thus, the term "host" may alternatively refer to animals such as mice, monkeys, dogs, pigs, rabbits, domesticated pigs (sows and hogs), ruminants, horses, poultry, cats, mice, bulls, dogs and the like, wherein the antibody, or antigen binding fragment, if necessary, is suitably engineered to achieve compatibility with the host.

As used herein, the term “polypeptide” means any polymer chain of amino acids. The terms "peptide" and "protein" are used interchangeably with the term "polypeptide" and also mean a polymer chain of amino acids. The term "polypeptide" includes natural and artificial proteins, protein fragments and polypeptide analogues of a protein sequence. The polypeptide may be monomeric or polymeric.

As used herein, the term “recovery” means the process of obtaining a chemical compound, such as a polypeptide, in a form substantially free of components naturally associated therewith, by isolation, for example, using protein purification techniques well known in the art.

As used herein, the terms “specific binding” or “specifically binding” with reference to the interaction of an antibody, protein or peptide with a second chemical entity mean that the interaction depends on the presence of a particular structure (for example, an “antigenic determinant” or “epitope”, the definition of which given below) on a chemical compound; for example, an antibody recognizes and binds to a specific protein structure rather than to any proteins. If an antibody is specific for epitope "A", the presence of a molecule containing epitope A (or free, unlabeled A) in a reaction mixture containing labeled "A" and the antibody will result in a decrease in the amount of labeled A bound to the antibody. If an epitope is mentioned herein, the anti-IgE antibody, or antigen binding fragment, of the invention is specific for said epitope.

As used herein, the term “antibody” broadly means any immunoglobulin (Ig) molecule consisting of four polypeptide chains, two heavy (H) chains and two light (L) chains, or any functional fragment, mutant, variant or derivative thereof , which retain at least some of the epitope-binding features of the Ig molecule, allowing them to specifically bind to IgE. Such mutant, variant or derivative antibody formats are known in the art and are described below. Non-limiting embodiments of such formats are known in the art and are described below. An antibody is said to be “capable of binding” a molecule (or epitope) if it is able to specifically react with the molecule (or epitope), resulting in binding of the molecule (or epitope) to the antibody.

As used herein, the term “monoclonal antibody” means a preparation of antibody molecules having the same heavy chain amino acid sequences and the same light chain amino acid sequences, or any functional fragment, mutant, variant or derivative thereof that retains at least the same epitope-binding features of the light chain as the Ig molecule, unlike “polyclonal” antibody preparations, which contain a mixture of different antibodies. Monoclonal antibodies can be produced using several known technologies, such as phage, bacterial, yeast or ribosomal display, as well as classical methods, for example, antibodies obtained from hybridomas (for example, an antibody secreted by a hybridoma obtained using hybridoma technology, such as standard methodology of Kohler and Milstein ((1975) Nature 256:495-497).

In a full-length antibody, each heavy chain consists of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region (CH). The heavy chain constant region consists of four domains—either CH1, hinge, CH2, and CH3 (γ, α, and δ heavy chains), or CH1, CH2, CH3, and CH4 (μ and ε heavy chains). Each light chain consists of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region (CL). The light chain constant region consists of a single domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs), which alternate with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Immunoglobulin molecules can be of any type (eg, IgG, IgE, IgM, IgD, IgA and IgY), class (eg, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass.

As used herein, the term “antigen-binding fragment” means one or more fragments or portions of an antibody that retain the ability to specifically bind an antigen (eg, IgE), or synthetic modifications of antibody fragments that retain the desired ability to bind an antigen. It has been shown that the antigen binding function of an antibody can be performed by fragments or some parts of the full-length antibody, or their modifications. Embodiments include bispecific, dual specificity, and multispecific formats that are capable of specifically binding to two or more different antigens or to multiple epitopes or discontinuous regions of epitopes of an antigen. Non-limiting examples of antigen binding fragments include (i) a Fab fragment, a monovalent fragment consisting of VL, VH, CL and CH1 domains; (ii) F(ab′) ₂ fragment, a divalent fragment containing two Fab fragments linked by a disulfide bond in the hinge region; (iii) Fd fragment consisting of VH and CH1 domains; (iv) Fv fragment consisting of the VL and VH domains of one arm of the antibody, (v) dAb fragment (Ward et al., (1989) Nature 341:544-546, Winter et al., PCT publication WO 90/05144 A1, the contents of which are incorporated by reference) containing one variable domain; (vi) a dedicated complementarity determining region (CDR), (vii) fusion products of antibody fragments, such as those of immunoglobulin nature, e.g. diabodies, scAbs, bispecific antibodies, tribodies, Fab-Fv, Fab-Fv-Fv, tribodies , (Fab-Fv) ₂ -Fc, and (viii) antibody moieties such as CDRs or antibody loops grafted onto non-immunoglobulin scaffolds such as fibronectin or leucine zippers (see Binz et al. (2005) Nat. Biotech 23:1257-1268, contents of publication incorporated by reference). In addition, although the two domains of the Fv fragment, VL and VH, are encoded by different genes, they can be joined recombinantly or otherwise using a synthetic or natural linker that allows them to exist as a single protein chain in which the VL and VH regions are paired linked to form monovalent molecules (known as single-chain Fv (scFv); see, for example, Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodies should also be included within the term "antigen binding fragment". Other forms of single chain antibodies, such as diabodies, are also covered by this term. Diabodies are divalent, bispecific antibodies in which the VH and VL domains are expressed as a single polypeptide chain, but using a linker that is too short to allow pairing between two domains on the same chain, causing the domains to be forced form pairs with complementary domains of the other chain, forming two antigen-binding sites (see, for example, Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, RJ, et al. (1994) Structure 2:1121-1123). Such binding antibody fragments are known in the art (Kontermann and Dubel eds., Antibody Engineering (2001) Springer-Verlag. New York. 790 pp. (ISBN 3-540-41354-5).

As used herein, the term “antibody construct” means a polypeptide comprising one or more antigen binding fragments of the invention linked to a linker polypeptide or immunoglobulin constant domain. Linker polypeptides contain two or more amino acid residues linked by peptide bonds and are used to link one or more antigen-binding moieties. Such linker polypeptides are well known in the art (see, for example, Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., et al. (1994) Structure 2:1121-1123). Immunoglobulin constant domain means a heavy or light chain constant domain, for example, human IgA, IgD, IgE, IgG or IgM constant domains. The amino acid sequences of the heavy chain and light chain constant domains are known in the art. Non-limiting examples of γ1 Ig heavy chain and λ and κ Ig light chain constant regions are shown in Tables 8 and 6, respectively.

In addition, the antibody, or an antigen-binding fragment thereof, may be part of a larger immunoadhesin molecule formed by covalent or non-covalent association of the antibody or antibody fragment with one or more other proteins or peptides. Examples of creating such immunoadhesin molecules include the use of the streptavidin core region to create a tetrameric scFv molecule (Kipriyanov, SM, et al. (1995) Human Antibodies and Hybridomas 6:93-101) and the use of a cysteine residue, a marker peptide, and a C-terminal polyhistidine marker to creating divalent and biotinylated scFv molecules (Kipriyanov, SM, et al. (1994) Mol. Immunol. 31:1047-1058). Antibody fragments, such as Fab and F(ab') ₂ fragments, can be prepared from whole antibodies by conventional methods such as papain or pepsin digestion, respectively, of whole antibodies. In addition, antibodies, antibody fragments and immunoadhesin molecules can be produced by standard recombinant DNA techniques as described herein.

As used herein, the term “isolated antibody” means an antibody that is substantially free from other antibodies having different antigen specificities (e.g., an isolated antibody that specifically binds IgE is substantially free from other antibodies that specifically bind antigens other than IgE ). However, an isolated antibody that specifically binds, for example, human IgE may have cross-reactivity with other antigens, such as IgE molecules from other species. In addition, the isolated antibody may be substantially free of other cellular material and/or chemical reagents.

The term “CDR-grafted antibody” refers to antibodies that contain heavy and light chain variable region sequences from one biological species, but in which the sequences of one or more of the VH and/or VL CDR regions are replaced by CDR sequences from another biological species, e.g. antibodies having human immunoglobulin heavy and light chain variable regions in which one or more of the human CDRs (eg, CDR3) are replaced with mouse CDR sequences.

Throughout this document, the terms “Kabat numbering,” “Kabat definitions,” and “Kabat markings” are used interchangeably. These terms, as recognized in the art, refer to a numbering system for amino acid residues that are more variable (i.e., hypervariable) than other amino acid residues in the variable regions of the heavy and light chains of an antibody, or an antigen binding fragment thereof (Kabat et al. ( 1971) Ann NY Acad Sci 190:382-391 and Kabat, E. A., et al (1991) Sequences of Proteins of Immunological Interest, fifth edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242 ). In the heavy chain variable region, the hypervariable region includes amino acid positions 31-35 (CDR-H1), residues 50-65 (CDR-H2) and residues 95-102 (CDR-H3) according to the Kabat numbering system. However, according to the Chothia system (Chothia et al., (1987) J. Mol. Biol., 196, 901-917 (1987)), the loop equivalent to CDR-H1 extends from residue 26 to residue 32. Thus Unless otherwise noted, when used herein, "CDR-H1" corresponds to residues 26-35, which is a combination of the Kabat numbering system and the Chothia topological loop definition. In the light chain variable region, the hypervariable region includes amino acid positions 24-34 for CDRL1, amino acid positions 50-56 for CDRL2, and amino acid positions 89-97 for CDRL3.

As used herein, the terms "acceptor" and "acceptor antibody" mean an antibody, or nucleotide sequence, that contains or encodes at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% of the amino acid sequences of one or more of the framework regions. In some embodiments, the term "acceptor" means the amino acid or nucleotide sequence of an antibody that constitutes, or encodes, constant region(s). In another embodiment, the term "acceptor" means an amino acid or nucleotide sequence of an antibody that represents, or encodes, one or more of framework regions and constant regions. In a specific embodiment, the term "acceptor" means an amino acid or nucleotide sequence of a human antibody that represents, or encodes, at least 80%, preferably at least 85%, at least 90%, at least 95%, of at least 98% or 100% of the amino acid sequences of one or more of the framework regions. In this embodiment, the acceptor may contain at least 1, at least 2, at least 3, at least 4, at least 5, or at least 10 amino acid residues that are not present at one or more specific positions in the antibody person. The acceptor framework region and/or acceptor constant region(s) may, for example, be derived from, or be derived from, a germline antibody gene, a mature antibody gene, a functional antibody (e.g., antibodies well known in the art, antibodies found in development, or antibodies commercially available).

As used herein, the term “CDR” means the complementarity determining region within the variable sequences of an antibody. There are three CDRs in each of the heavy chain and light chain variable regions, which are designated CDRH1, CDRH2 and CDRH3 in the case of the heavy chain CDR, and CDRL1, CDRL2 and CDRL3 in the case of the light chain CDR. As used herein, the term “set of CDRs” means a group of three CDRs that are present in a single variable region capable of binding an antigen. The exact boundaries of these CDRs are defined differently according to different systems. The system described by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides a unique residue numbering system applicable to any antibody variable region, but also allows precise delineation of the residues that make up the three CDRs. These CDRs may be called Kabat CDRs. Chothia and co-workers (Chothia & Lesk, J. Mol. Biol. 196:901-917 (1987) and Chothia et al., Nature 342:877 -883 (1989)) found that some subfragments within the Kabat CDR adopt almost identical peptide backbone conformations despite large differences at the amino acid sequence level. These subfragments were designated L1, L2 and L3 or H1, H2 and H3, where "L " and "H" stand for light chain regions and heavy chain regions, respectively. These regions can be called Chothia CDRs and have boundaries that overlap with the Kabat CDR. Other boundaries defining CDRs that overlap with the Kabat CDR have been described by Padlan (FASEB J. 9:133-139 (1995)) and MacCallum (J Mol Biol 262(5):732-45 (1996)). Other CDR boundary definitions may not strictly follow one of the systems described above, but will nevertheless have overlap with the Kabat CDR, although they may be shortened or lengthened based on prediction or experimental results indicating that particular residues or groups residues, or even entire CDRs do not have a significant effect on antigen binding. The methods described herein may use CDRs whose boundaries are defined in accordance with one of the described systems, although preferred embodiments use CDRs whose boundaries are defined in accordance with the Kabat or Chothia system, or a combination thereof.

As used herein, the term “canonical” residue refers to a residue in a CDR or framework that defines a particular canonical CDR structure according to the system of Chothia et al. (J. Mol. Biol. 196:901-907 (1987); Chothia et al., J. Mol. Biol. 227:799 (1992), both incorporated herein by reference). According to Chothia et al., critical portions of the CDRs of many antibodies have nearly identical peptide backbone conformations, despite large differences at the amino acid sequence level. Each canonical structure essentially defines a set of rotation angles for the peptide backbone so that a contiguous segment of amino acid residues forms a loop.

As used herein, the terms “donor” and “donor antibody” refer to an antibody providing one or more CDRs. In a preferred embodiment, the donor antibody is an antibody from a biological species other than the species from which the antibody framework regions were derived or derived. In the context of a humanized antibody, the term “donor antibody” means a non-human antibody that provides one or more CDRs.

As used herein, the term “framework” or “framework sequence” refers to the remaining variable region sequences minus the CDR regions. Since the exact CDR sequences may be determined according to different systems, the definition of the framework sequence may accordingly have different interpretations. The six CDR regions (light chain CDR-L1, -L2 and -L3 and heavy chain CDR-H1, -H2 and -H3) also divide the framework regions in the light chain and heavy chain into four sub-regions (FR1, FR2, FR3 and FR4) in each chain, with CDR1 located between FR1 and FR2, CDR2 between FR2 and FR3, and CDR3 between FR3 and FR4. Without designating specific subregions as FR1, FR2, FR3 or FR4, the framework region, as noted by others, represents the combined FRs in the variable region of one natural immunoglobulin chain. As used herein, “FR region” means one of the four sub-regions, and “FR regions” means two or more of the four sub-regions constituting the framework region.

As used herein, the term “germline antibody gene” or “gene fragment” means an immunoglobulin sequence encoded in non-lymphoid cells that have not undergone the maturation process leading to genetic rearrangement and mutation to express a particular immunoglobulin. See, for example, Shapiro et al., Crit. Rev. Immunol. 22(3):183-200 (2002); Marchalonis et al., Adv Exp Med Biol. 484:13-30 (2001). One of the advantages provided by various embodiments of the present invention is based on the recognition that germline antibody genes are more likely than mature antibody genes to retain the essential amino acid sequence structures characteristic of individuals within a species, and therefore will be less likely to be recognized as coming from an extraneous source when used therapeutically for these species.

As used herein, the term “key” residues refers to certain residues in the variable region that have a greater impact on the binding specificity and/or affinity of an antibody, particularly a humanized antibody. A key residue includes, but is not limited to, one or more of the following: a residue adjacent to a CDR, a potential glycosylation site (can be either an N- or O-glycosylation site), a rare residue, a residue capable of interacting with an antigen, a residue capable of interact with a CDR, a canonical residue, a contact residue between a heavy chain variable region and a light chain variable region, a residue in the Vernier zone, and a residue in the overlap region between CDR1 of the heavy chain variable region as defined by Chothia and the first heavy chain framework region as defined by Kabat.

The term “humanized antibody” generally refers to antibodies that contain heavy and light chain variable region sequences from a non-human species (eg, rabbit, mouse, etc.) but in which at least a portion of the VH sequence and /or VL has been modified to become more “human-like,” that is, more similar to human germline variable sequences. One type of humanized antibody is a CDR-grafted antibody, in which human CDR sequences are inserted into non-human VH and VL sequences to replace the corresponding non-human CDR sequences. Another type of humanized antibody is a CDR-grafted antibody, in which at least one non-human CDR is embedded in a human immunoglobulin backbone. The present invention generally focuses on the latter option.

In particular, as used herein, the term “humanized antibody” means an antibody, or a variant, derivative, analogue, or fragment thereof, that immunospecifically binds to an antigen of interest and that contains a framework (FR) region having substantially the amino acid sequence of a human antibody. and a complementarity determining region (CDR) having essentially the amino acid sequence of a non-human antibody. As used herein, the term “substantially” in the context of a CDR refers to a CDR having an amino acid sequence of at least 50, 55, 60, 65, 70, 75 or 80%, preferably at least 85%, at least 90 %, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of the non-human antibody CDR. In one embodiment, the humanized antibody comprises a CDR region having one or more (eg, 1, 2, 3, or 4) amino acid substitutions, additions, and/or deletions compared to the CDR of the non-human antibody. In addition, a non-human CDR may be genetically modified to become more “human-like” or compatible with the human body using known methods. A humanized antibody contains substantially all of at least one and typically two variable domains (Fab, Fab', F(ab') ₂ , F(ab')c, Fv), all or substantially all of the CDR regions correspond to CDR regions of a non-human immunoglobulin (ie, donor antibody), and all or substantially all of the framework regions are human immunoglobulin consensus sequence framework regions. Preferably, the humanized antibody also contains at least a portion of the constant region (Fc) of an immunoglobulin, typically human immunoglobulin. In some embodiments, the humanized antibody contains both a light chain and at least a heavy chain variable domain. The antibody may also contain CH1, hinge, CH2 and CH3, or CH1, CH2, CH3 and CH4 of the heavy chain. In some embodiments, the humanized antibody contains only a humanized light chain. In some embodiments, the humanized antibody contains only the humanized heavy chain. In specific embodiments, the humanized antibody comprises only a humanized light chain variable domain and/or a humanized heavy chain. Although some of the mutations described herein may not be conventionally humanizing, this is no reason to not consider the anti-IgE antibody, or antigen binding fragment, of the invention to be “humanized.”

The humanized antibody can be selected from any class of immunoglobulins, including IgY, IgM, IgG, IgD, IgA, and IgE, and any isotype, including, but not limited to, IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4. A humanized antibody may contain sequences from more than one class or isotype of immunoglobulin, and specific constant domains may be selected to optimize desired effector functions using methods well known in the art.

The framework and CDR regions of a humanized antibody do not need to correspond exactly to the original sequences, for example, the donor antibody CDR or consensus framework can be mutagenized by substitution, insertion and/or deletion of at least one amino acid residue such that the CDR or framework residue at a given site does not exactly match either the donor antibody or the consensus scaffold. In a preferred embodiment, such mutations, however, will not be extensive. Typically at least 50, 55, 60, 65, 70, 75 or 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95%, 98% or 99% residues of the humanized antibody will correspond to residues in the original FR and CDR sequences. In one embodiment, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid substitutions, additions, and/or deletions may occur in the humanized antibody relative to the parent sequences FR and CDR (eg, compared to omalizumab or omalizumab Fab sequences). As used herein, the term “consensus framework” means a framework region in an immunoglobulin consensus sequence. As used herein, the term “immunoglobulin consensus sequence” means a sequence formed from the most frequently occurring amino acids (or nucleotides) in a family of related immunoglobulin sequences (see, for example, Winnaker, From Genes to Clones (Verlagsgesellschaft, Weinheim, Germany 1987). In the family immunoglobulins, each position in the consensus sequence is occupied by the amino acid that occurs most frequently at that position in a given family.If two amino acids occur with the same frequency, either one can be included in the consensus sequence.

As used herein, the term “Vernier zone” refers to a subset of framework residues that can regulate the structure of the CDR and fine-tune it to match the antigen, as described by Foote and Winter (1992, J. Mol. Biol. 224:487-499, contents included this document by reference). The Vernier zone remnants form a layer underneath the CDR and can influence the structure of the CDR and the affinity of the antibody.

As used herein, the term “neutralization” means neutralization of the biological activity of IgE when an anti-IgE antibody, or antigen binding fragment, of the invention described herein specifically binds the IgE protein. Neutralization may be the result of different binding patterns of the specified antibody to IgE. Preferably, the neutralizing antibody is an antibody whose binding to IgE results in neutralization of the biological activity of the IgE. Preferably, the neutralizing binding protein binds IgE and reduces the biological activity of IgE by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 80%, 85% or more. Neutralization of IgE biological activity by a neutralizing antibody can be assessed by measuring one or more of the IgE biological activity indicators described herein.

As used herein, the term “neutralizing monoclonal antibody” means a preparation of antibody molecules that, when bound to IgE, are capable of inhibiting or reducing the biological activity of IgE, either partially or completely.

As used herein, the terms “reduce,” “attenuate,” and the like mean a reduction or reduction in the severity of a symptom or condition caused by elevated serum levels of IgE.

The terms "epitope" or "antigenic determinant" cover any polypeptide determinant that is capable of specifically binding to an immunoglobulin or T-cell receptor. In certain embodiments, antigenic determinants include reactive surface moieties of molecules such as amino acids, sugar side chains, phosphoryl, or sulfonyl, and in certain embodiments they may have specific three-dimensional structure characteristics and/or specific charge characteristics. An epitope is the region of an antigen that is bound by an antibody. In specific embodiments, an antibody is said to specifically bind an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and/or macromolecules.

As used herein, the term " _kon " means the rate constant of association of an antibody with an antigen to form an antibody/antigen complex, as is known in the art.

As used herein, the term " _koff " means the rate constant for the dissociation of an antibody from an antibody/antigen complex, as is known in the art.

As used herein, the term “k _d ” or “k _D ” means the dissociation constant for a particular antibody-antigen interaction as known in the art.

The strength, or affinity, of an interaction in immunological binding can be expressed in terms of the dissociation constant (k _D or k _d ) of the interaction, with a lower value of k _d corresponding to greater or higher affinity. Immunological binding scores of selected polypeptides can be calculated using methods well known in the art. One such method involves measuring the rates of formation and dissociation of the antigen-binding site/antigen complex, with these rates depending on the concentrations of the complex partners, the affinity of the interaction, and geometric parameters, which equally influence the rates of both processes. Thus, both the “association rate constant” (“k _on ”) and the “dissociation rate constant” (“k _off ”) can be determined by calculating the concentrations and actual rates of association and dissociation. (Nature 361:186-87 (1993)). The ratio k _off /k _on allows us to discard all parameters not related to affinity and is equal to the dissociation constant k _d . Davies et al. (1990) Annual Rev Biochem 59:439–473.

The term "antibody conjugate" means a binding protein, such as an antibody or antibody fragment, or a binding fragment thereof, chemically linked to a second chemical compound, such as a therapeutic or cytotoxic agent. The term “agent” is used herein to refer to a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract derived from biological materials.

As used herein, the terms “crystal” and “crystallized” refer to an antibody, or an antigen-binding fragment thereof, in the form of a crystal. Crystals are a form of solid state of matter, distinct from other forms such as amorphous solid state or liquid crystalline state. Crystals are composed of regular, repeating three-dimensional lattices of atoms, ions, molecules (eg, proteins such as antibodies), or molecular aggregates (eg, antigen/antibody complexes). These three-dimensional lattices are arranged according to certain mathematical relationships that are well known in the art. A fundamental unit or building block that is repeated in a crystal is called an asymmetric unit. Repeating asymmetric units in a spatial configuration that corresponds to a given well-defined crystallographic symmetry create the "unit cell" of the crystal. Repeating unit cells due to regular transfers (translations) in all three dimensions create a crystal. See Giege, R. and Ducruix, A. Barrett, Crystallization of Nucleic Acids and Proteins, a Practical Approach, 2nd ed., pp. 201-16, Oxford University Press, New York, N.Y., (1999).

As used herein, the term "polynucleotide" means a polymeric form of two or more nucleotides, either ribonucleotides, or deoxynucleotides, or modified forms of each type of nucleotide. The term includes single- and double-stranded forms of DNA, but preferably means double-stranded DNA.

As used herein, the term “isolated polynucleotide” means a polynucleotide (e.g., of genomic, cDNA, or synthetic origin, or a combination thereof) that, by virtue of its name, “isolated polynucleotide”, is not related to all or part of the polynucleotide to which the “isolated polynucleotide” is polynucleotide" occurs in nature; functionally associated with a polynucleotide to which it is not naturally associated; or does not exist in nature as part of a larger sequence.

As used herein, the term “vector” means a nucleic acid molecule capable of carrying another nucleic acid to which it has been linked. One type of vector is a "plasmid", which is a circular double-stranded loop of DNA into which additional DNA segments can be ligated. Another type of vector is a viral vector, in which case additional DNA segments can be ligated into the viral genome. Some vectors are capable of autonomous replication in the host cell into which they are introduced (eg, bacterial vectors having a bacterial origin of replication and mammalian episomal vectors). Other vectors (eg, non-episomal mammalian vectors) can integrate into the host cell genome upon introduction into the host cell and thus replicate along with the host genome. In addition, some vectors are capable of controlling the expression of genes to which they are functionally linked. Such vectors are referred to herein as “recombinant expression vectors” (or simply, “expression vectors”). Typically, expression vectors used in recombinant DNA techniques are often in the form of a plasmid. In this specification, the terms “plasmid” and “vector” may be used interchangeably because plasmid is the most commonly used form of vector. However, the invention is intended to cover other forms of expression vectors, such as viral vectors (eg, replication-defective retroviruses, adenoviruses and adeno-associated viruses) that perform equivalent functions.

The term "operably linked" refers to the adjacent arrangement of components in which the described components are in a relationship that allows them to function in the manner intended for them. The control sequence "operably linked" to the coding sequence is ligated such that expression of the coding sequence is achieved under conditions determined by the control sequences. "Operably linked" sequences include both expression control sequences that are located in close proximity to the gene of interest and expression control sequences that act in trans, or at a distance, to control the gene of interest. As used herein, the term “expression control sequence” refers to polynucleotide sequences that are necessary to effect the expression and processing of the coding sequences to which they are ligated. Expression control sequences include corresponding transcription initiation, termination, promoter and enhancer sequences; signals for efficient RNA processing, such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that increase translation efficiency (ie, Kozak consensus sequence); sequences that increase protein stability, and, if necessary, sequences that increase protein secretion. The nature of such control sequences varies depending on the host organism; in prokaryotes, such control sequences typically include a promoter, a ribosome binding site, and a transcription termination sequence; In eukaryotes, such control sequences typically include promoters and transcription termination sequences. The term “control sequences” includes components whose presence is essential for expression and processing, and may also include additional components whose presence is beneficial, such as leader sequences and fusion partner sequences.

As used herein, the term “transformation” means any process by which exogenous DNA enters a host cell. The transformation can occur under natural or artificial conditions, using various methods well known in the art. The transformation can be based on any known method of introducing foreign nucleotide sequences into a prokaryotic or eukaryotic host cell. The method is selected based on the host cell being transformed and may include, but is not limited to, viral infection, electroporation, lipofection, and particle bombardment. Such “transformed” cells include stably transformed cells in which the introduced DNA is capable of replication, either as an autonomously replicating plasmid or as part of a host chromosome. They also include cells that transiently express the introduced DNA or RNA for limited periods of time.

As used herein, the term “recombinant host cell” (or simply “host cell”) means a cell into which exogenous DNA has been introduced. It should be understood that such terms are intended to refer not only to the specific cell referred to, but also to the progeny of such cell. Since certain modifications may occur in subsequent generations due to either mutation or environmental influences, such progeny may not actually be identical to the original cell, however, it still falls within the definition of the term “host cell” as used herein. Preferred host cells include prokaryotic and eukaryotic cells selected from any kingdom of living organisms. Preferred eukaryotic cells include protozoan, fungal, plant and animal cells. Most preferably, host cells include, but are not limited to, a prokaryotic cell line of E. coli; mammalian cell lines CHO, HEK 293 and COS; insect cell line Sf9 and fungal cells Saccharomyces cerevisiae.

Standard methods for recombinant DNA production, oligonucleotide synthesis, and tissue culture and transformation (eg, electroporation, lipofection) can be used. Enzymatic reactions and purification methods can be used in accordance with the manufacturer's instructions, as is generally accepted in the art or as described herein. The above methods and procedures generally can be used in a manner generally known in the art and as described in the various general and specialized literature cited and discussed throughout this specification. See, for example, Sambrook et al. Molecular Cloning: A Laboratory Manual (2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), the contents of which are incorporated herein by reference for all purposes.

As used herein, the term “effective amount” means an amount of therapeutic agent that is sufficient to attenuate or reduce the severity and/or duration of a disease, or one or more symptoms thereof, prevent the progression of a disease, cause regression of a disease, prevent relapse, development, occurrence or progression of one or more symptoms associated with a disease, detection of a disease, or enhancement or improvement of the prophylactic or therapeutic effect(s) of another drug (eg, a prophylactic or therapeutic agent).

A map of specific regions or epitopes of the human IgE protein described herein can be determined by any suitable epitope mapping method known in the art in combination with any of the antibodies provided by the present invention. Examples of such methods include screening IgE-derived peptides of varying lengths for binding to an antibody of the present invention, wherein the smallest fragment that specifically binds to the antibody contains an epitope sequence recognized by the antibody. IgE peptides can be produced by synthesis or by proteolytic cleavage of the IgE protein. Peptides that bind the antibody can be identified, for example, using mass spectroscopic analysis. In another example, NMR spectroscopy or X-ray crystallography can be used to identify an epitope bound by an antibody of the present invention. Crystallization and X-ray crystallography methods are preferred for determining the structure of IgE and the epitope on IgE to which the anti-IgE antibody, or antigen binding fragment, of the invention binds.

Antibodies used in the invention can be obtained by methods for producing antibodies from single lymphocytes by cloning and expressing cDNAs of immunoglobulin variable regions obtained from single lymphocytes selected on the basis of production of specific antibodies, for example, methods described in Babcook, J. et al., 1996 ,Proc. Natl. Acad. Sci. USA 93(15):7843-7848l; WO92/02551; WO2004/051268 and international patent application number WO2004/106377. Screening for antibodies can be performed using assays to measure binding to human IgE and/or assays to measure the ability to block the binding of IgE to its natural receptor. An example of a binding assay is ELISA.

Humanized antibodies (which include CDR-grafted antibodies) are antibody molecules having one or more complementarity determining regions (CDRs) of an immunoglobulin from a non-human species (eg, rabbit or mouse) and a framework region from a human immunoglobulin molecule (see eg US 5585089; WO91/09967). It should be understood that it may be necessary to transfer only specificity-determining residues of CDR regions rather than entire CDRs (see, for example, Kashmiri et al., 2005, Methods, 36, 25-34). Humanized antibodies may optionally also contain one or more framework residues from an immunoglobulin of a non-human species from which the CDRs were derived. The latter are often called donor residues. The antibody molecules of the present invention preferably have a binding affinity ( _KD ) of less than 2 nM. Affinity can be measured by any suitable method known in the art, including BIAcore, as described in the Examples section of this document (see Example 6), using isolated natural or recombinant IgE, or a suitable fusion protein/polypeptide.

The affinity of an antibody, or antigen binding fragment, of the present invention, as well as the extent to which a binding compound (such as an antibody) inhibits binding, can be determined by one of ordinary skill in the art using conventional methods, such as those described in Scatchard et al. (Ann. KY. Acad. Sci. 51:660-672 (1949)), or the surface plasmon resonance (SPR) method using systems such as BIAcore. In surface plasmon resonance, target molecules are immobilized on a solid phase and exposed to ligands in a mobile phase flowing through a flow cell. If ligand binding to the immobilized target occurs, the local refractive index changes, resulting in a change in the SPR angle, which can be monitored in real time by detecting changes in reflected light intensity. The rates of change of the SPR signal can be analyzed to determine the apparent rate constants for the association and dissociation phases of the binding reaction. The ratio of these quantities gives the apparent equilibrium constant (affinity) (see, for example, Wolff et al., Cancer Res. 53:2560-65 (1993)).

It should be understood that the affinity of the antibodies of the present invention can be changed by any suitable method known in the art. Thus, the present invention also provides variants of the antibody molecules of the present invention that have improved affinity for IgE. Such variants can be generated using a variety of affinity maturation techniques, including CDR mutations (Yang et al., J. Mol. Biol., 254, 392-403, 1995), chain shuffling (Marks et al., Bio/Technology, 10, 779-783, 1992), the use of E. coli mutator strains (Low et al., J. Mol. Biol., 250, 359-368, 1996), DNA shuffling (Patten et al., Curr. Opin. Biotechnol., 8, 724-733, 1997), phage display (Thompson et al., J. Mol. Biol., 256, 77-88, 1996) and sexual simulation PCR (Crameri et al., Nature, 391 , 288-291, 1998). The article by Vaughan et al. (above) describes these affinity maturation methods.

Humanized antibodies and antigen-binding fragments

In one aspect of the present invention, humanized anti-IgE monoclonal antibodies and antigen binding fragments are provided. Humanized antibodies are antibodies in which the heavy and/or light chain contains one or more CDRs (including, if necessary, one or more modified CDRs) from a donor antibody (for example, a non-human antibody, such as a mouse or rabbit monoclonal antibody ) grafted onto the heavy and/or light chain variable region framework of an acceptor antibody (eg, a human antibody). For review see Vaughan et al, Nature Biotechnology, 16, 535-539, 1998.

In one embodiment, instead of transferring the entire CDR region to the human antibody framework, only one or more specificity-determining residues from any of the CDRs described above are transferred herein (see, for example, Kashmiri et al., 2005, Methods, 36, 25- 34). In one embodiment, only the specificity-determining residues from one or more of the CDRs described herein are transferred to the human antibody framework. In another embodiment, only the specificity-determining residues from each of the CDRs described herein are transferred to the human antibody framework.

When grafting CDRs or specificity-determining residues, any suitable variable region acceptor framework sequence can be used, taking into account the class/type of donor antibody from which the CDRs are derived, including mouse, rabbit, primate and human antibody frameworks.

Preferably, the humanized antibody of the present invention has a variable domain containing human acceptor framework regions, as well as one or more CDRs specifically described herein. Thus, in one embodiment, a humanized monoclonal antibody is provided that binds human IgE, wherein the variable domain contains human acceptor framework regions (with optional mutations described herein) and non-human donor CDRs.

The construction of CDR-grafted antibodies is generally described in European Patent Application EP-A-0239400, which discloses a method for grafting CDRs of a murine monoclonal antibody onto human immunoglobulin variable domain frameworks by site-directed mutagenesis using long oligonucleotides, the contents of which are included in this document. CDR regions determine the antigen-binding specificity of antibodies and are relatively short peptide sequences located on the framework regions of the variable domains.

The earliest work on the humanization of monoclonal antibodies by grafting CDRs was carried out on monoclonal antibodies recognizing synthetic antigens, such as NP. However, examples in which a mouse monoclonal antibody recognizing lysozyme and a rat monoclonal antibody recognizing an antigen on human T cells were humanized by grafting CDRs were described by Verhoeyen et al. (Science, 239, 1534-1536, 1988) and Riechmann et al. (Nature, 332, 323-324, 1988), respectively. Antibody humanization is accomplished by grafting the CDR of a non-human antibody, such as a mouse, rat, goat, or rabbit antibody, onto a “similar” scaffold (acceptor) of a human antibody and selecting a minimum number of key scaffold residues (back mutations) that are manually selected from the donor monoclonal antibodies and are inserted into the acceptor framework of a human antibody to preserve the original CDR conformation. Such methods are known in the art and include those described in Jones et al., Nature 321:522 (1986); Verhoeyen et al., Science 239:1534 (1988)), Sims et al., J. Immunol. 151:2296 (1993); Chothia and Lesk, J. Mol. Biol. 196:901 (1987), Carter et al., Proc. Natl. Acad. Sci. U.S.A. 89:4285 (1992); Presta et al., J. Immunol. 151:2623 (1993), Padlan, Molecular Immunology 28(4/5):489-498 (1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994); Roguska. et al., PNAS 91:969-973 (1994); PCT publications WO 91/09967, PCT/: US98/16280, US96/18978, US91/09630, US91/05939, US94/01234, GB89/01334, GB91/01134, GB92/01755; WO90/14443, WO90/14424, WO90/14430, EP 229246, EP 592.106; EP 519,596, EP 239,400, US Patent Nos. 5565332, 5723323, 5976862, 5824514, 5817483, 5814476, 5763192, 5723323, 5766886, 5714352, 620402 3, 6180370, 5693762, 5530101, 5585089, 5225539; 4816567, the contents of which are included herein.

The classification of human germline heavy and light chain variable sequence subfamilies can be derived from the germline subgroup designations according to the Kabat system: VH1, VH2, VH3, VH4, VH5, VH6 or VH7 for a particular VH sequence and JH1, JH2, JH3, JH4 , JH5 and JH6 for a specific heavy chain variable region connecting group for framework 4; VK1, VK2, VK3, VK4, VK5 or VK6 for a specific VL kappa sequence for framework 1, 2 and 3, and JK1, JK2, JK3, JK4 or JK5 for a specific kappa connecting group for framework 4; or VL1, VL2, VL3, VL4, VL5, VL6, VL7, VL8, VL9 or VL10 for a specific VL lambda sequence for scaffold 1, 2 and 3, and JL1, JL2, JL3 or JL7 for a specific lambda connecting group for scaffold 4.

The constant region domains of the antibody molecule of the present invention, if present, may be selected based on the intended function of the antibody molecule and, in particular, the effector functions that may be required. For example, the constant region domains may be human IgA, IgD, IgE, IgG, or IgM domains. In particular embodiments, human IgG constant region domains, particularly those of the IgG1 and IgG3 isotypes, may be used if the antibody molecule is intended for therapeutic use and the effector functions of the antibody are desired. Alternatively, the IgG2 and IgG4 isotypes can be used if the antibody molecule is intended for therapeutic purposes and the effector functions of the antibody are not required. It should be understood that sequence variants of these constant region domains may also be used. For example, IgG4 molecules can be used in which the serine at position 241 has been replaced by proline, as described in Angal et al., Molecular Immunology, 1993, 30 (1), 105-108. One of ordinary skill in the art also understands that antibodies can undergo various post-translational modifications. The type and extent of such modifications often depend on the host cell line used to express the antibody, as well as the culture conditions. Such modifications may include variations in glycosylation, methionine oxidation, diketopiperazine formation, aspartate isomerization, and asparagine deamidation. A common modification is the loss of a carboxy-terminal basic residue (such as lysine or arginine) as a result of the action of carboxypeptidases (as described in Harris, R. J. Journal of Chromatography 705:129-134, 1995). Accordingly, the C-terminal lysine residue of the antibody heavy chain may be absent.

CDRs and modifications of the human immunoglobulin framework region

Riechmann et al. found that transfer of CDR alone (as described by Kabat et al. (supra) and Wu et al., J. Exp. Med., 132, 211-250, 1970) was not sufficient to provide satisfactory ability to antigen binding in the CDR-grafted product. It was found that a number of framework residues needed to be modified to match those of the donor framework region. Suggested criteria for selecting frame residues that need to be modified are given in international patent application WO90/07861, the contents of which are included herein.

Grafting non-human CDRs onto a human variable domain backbone is most likely to result in retention of the correct spatial orientation of the CDRs if the human variable domain backbone adopts the same or similar conformation as the non-human variable domain backbone from which the CDRs were taken. This is achieved by obtaining human variable domains from human antibodies whose framework sequences have a high degree of sequence identity to the non-human variable domain framework sequences from which the CDRs were derived. As described above, the heavy and light chain variable region framework regions can be taken from the same or different human antibody sequences. The human antibody sequences may be natural human antibody sequences or may be consensus sequences of several human antibodies. See Kettleborough et al, Protein Engineering 4:773 (1991); Kolbinger et al., Protein Engineering 6:971 (1993) and Carter et al., WO92/22653.

Having identified the complementarity determining regions of the non-human donor immunoglobulin and the corresponding acceptor human immunoglobulins, the next step is to determine which, if necessary, residues of these components should be replaced to optimize the properties of the resulting humanized antibody. In general, substitution of human amino acid residues with non-human amino acid residues should be kept to a minimum because the introduction of non-human amino acid residues increases the risk that the antibody will induce an anti-donor antibody (HADA) immune response in the human. Art-recognized methods for determining the immune response can be used to monitor the HADA response in a specific host or in clinical trials. It is possible to evaluate immunogenicity in hosts treated with humanized antibodies at the start of the study and during administration of the therapeutic agent. The HADA response is determined, for example, by detecting antibodies to the humanized therapeutic agent in host serum samples by a method known in the art, including surface plasmon resonance technology (BIACORE) and/or solid-phase ELISA.

The choice of amino acid residues to be replaced (also referred to herein as “mutations”) is determined in part by the results of computer modeling. This document describes computer devices and software for creating three-dimensional images of immunoglobulin molecules. As a rule, molecular models are created starting from known structures of immunoglobulin chains or their domains. Modeled chains are compared for similarity of amino acid sequences to chains or domains of known three-dimensional structures, and the chains or domains exhibiting the greatest sequence similarity are selected as starting points for constructing the molecular model. Chains or domains having at least 50% sequence identity are selected for modeling, and preferably those having at least 60%, 70%, 80%, 90% or more sequence identity are selected for modeling. Known starting structures are modified to account for differences between the actual amino acids in the immunoglobulin chains or domains being modeled and those present in the starting structure. The modified structures are then assembled into a combined immunoglobulin. Finally, the model is refined by minimizing the energy and checking that all atoms are at appropriate distances from each other and that the bond lengths and angles are within chemically acceptable limits.

The selection of amino acid residues for substitution may also be determined in part by the results of studies of the characteristics of amino acids at specific locations, or by empirical observation of the effects of substitution or mutagenesis of specific amino acids. For example, if there are differences between a donor variable region framework residue and a selected human variable region framework residue, the human framework amino acid generally should be replaced with an equivalent framework amino acid from the donor antibody if it is believed that the amino acid:

(1) directly non-covalently binds antigen,

(2) is adjacent to the CDR area,

(3) otherwise interacts with the CDR region (eg, is within 3-6 angstroms of the CDR region as determined by computer modeling), or

(4) is located in the VL-VH contact area.

Residues that “directly non-covalently bind antigen” include amino acids at positions in framework regions that are likely to directly interact with amino acids of the antigen in accordance with known chemical forces such as hydrogen bonding, van der Waals interactions, hydrophobic interactions, and the like . The CDRs and framework regions follow the system of Kabat et al. or Chothia et al., supra. If the framework residues defined by the system of Kabat et al., supra, are structural loop residues according to the system of Chothia et al., supra, then the amino acids present in the donor antibody can be selected for substitutive introduction into the humanized antibody. Residues that are “adjacent to the CDR region” include amino acid residues at positions in close proximity to one or more CDRs in the basic chain sequence of a humanized immunoglobulin, for example, at positions in close proximity to a CDR defined by the Kabat system, or CDR defined according to the Chothia system (see, for example, Chothia and Lesk 1MB 196: 901 (1987)). There is a strong possibility that these amino acids interact with amino acids in the CDR and, if selected from the acceptor, will cause distortion of the donor CDR and lead to a decrease in affinity. In addition, amino acids located in close proximity may directly interact with the antigen (Amit et al., Science, 233:747 (1986), the contents of the publication are incorporated herein by reference) and the selection of such amino acids from the donor may be desirable to preserve all contacts with the antigen, which provide the affinity of the original antibody. As described herein, substitutions/mutations can also be made in the FR sequences to increase the affinity of the anti-IgE antibody, or antigen binding fragment, of the invention for IgE (and/or to expand its interaction region or epitope to Cε2 in IgE).

Residues that “otherwise interact with the CDR region” include residues that, as determined by secondary structure analysis, are in a spatial orientation that may influence the CDR region. In one embodiment, residues that "otherwise interact with the CDR region" are identified by analyzing a three-dimensional model of the donor immunoglobulin (eg, a computer model). The 3D model of the original donor antibody typically shows that certain amino acids outside the CDR are located close to the CDR, and there is a high probability of them interacting with amino acids in the CDR through hydrogen bonding, van der Waals interactions, hydrophobic interactions, and so on. . For such amino acid positions, it is preferable to select a donor immunoglobulin amino acid rather than an acceptor immunoglobulin amino acid. Amino acids that meet this criterion will generally have a side chain atom within about 3 angstroms (A) of some atom in the CDR regions and must contain an atom that could interact with the CDR atoms in accordance with known chemical forces such as those which are listed above. In the case of atoms that can form a hydrogen bond, the measured distance is 3 Å between their nuclei, but for atoms that do not form a bond, the measured distance is 3 Å between their van der Waals surfaces. Thus, in the latter case, the nuclei must be within 6 Å of a distance (3 Å plus the sum of the van der Waals radii) to be considered capable of interaction. In many cases the nuclei will be 4 or 5 to 6 Å apart. When determining whether an amino acid can interact with CDR regions, it is preferable not to consider the last 8 amino acids of the heavy chain CDR2 as part of the CDR, since from a structural standpoint these 8 amino acids are more of a scaffold.

Amino acids that are capable of interacting with amino acids in the CDR regions (or FR regions) can be identified in another way. The solvent accessible surface area of each backbone amino acid is calculated in two ways: (1) in the intact antibody and (2) in a hypothetical molecule that is an antibody with the CDR regions removed. A significant difference between these values of about 10 square angstroms or more indicates that solvent access to the framework amino acid is at least partially blocked by the CDR regions and, therefore, that the amino acid is in contact with the CDR regions. The solvent accessible surface area of an amino acid can be calculated from a three-dimensional model of the antibody using algorithms known in the art (eg, Connolly, J. Appl. Cryst. 16:548 (1983) and Lee and Richards, J. Mol. Biol. 55 :379 (1971), the contents of both publications are incorporated herein by reference). Backbone amino acids can also sometimes interact with CDR regions indirectly by influencing the conformation of another backbone amino acid, which in turn contacts the CDR regions.

In many antibodies, specific amino acids at certain positions in the framework region are known to interact with CDR regions (Chothia and Lesk, supra, Chothia et al., supra, and Tramontano et al., J. Mol. Biol. 215:175 (1990), the contents of all publications are incorporated herein by reference). In particular, it is known that in many antibodies, amino acids at positions 2, 48, 64 and 71 in the light chain, and 71 and 94 in the heavy chain (Kabat numbering) are capable of interacting with CDR regions. It is also possible that amino acids at positions 35 in the light chain and 93 and 103 in the heavy chain interact with the CDR regions. In all of these listed positions, it is preferable to select the donor amino acid rather than the acceptor amino acid (if different) for the humanized immunoglobulin. On the other hand, certain residues capable of interacting with the CDR region, such as the first 5 amino acids of the light chain, can sometimes be selected from the acceptor immunoglobulin without losing the affinity of the humanized immunoglobulin.

Residues that are “at the VL-VH interface” or “spacer residues” include residues at the interface between VL and VH as defined by, for example, Novotny and Haber, Proc. Natl. Acad. Sci. USA, 82:4592-66 (1985) or Chothia et al., supra. In general, unusual spacer residues should be retained in the humanized antibody if they are different from those in the human framework regions.

Typically, one or more of the amino acids meeting the above criteria are replaced. In some embodiments, all or most of the amino acids meeting the above criteria are replaced. Sometimes there is some uncertainty as to whether a particular amino acid satisfies the above criteria, then alternative immunoglobulins are produced, one of which has that particular substitution and the other does not. The alternative immunoglobulins thus obtained can be tested in any of the assays described herein for the presence of the desired activity and the preferred immunoglobulin selected.

Typically, the CDR regions in humanized antibodies are substantially identical and, most often, identical to the corresponding CDR regions of the donor antibody. Although not generally desirable, it is sometimes possible to make one or more conservative amino acid substitutions of CDR residues without significantly affecting the binding affinity of the resulting humanized immunoglobulin. Conservative or similar substitutions are substitutions such as, for example, replacing leucine with isoleucine or valine. Other amino acids that can often substitute for one another include, but are not limited to:

phenylalanine, tyrosine and tryptophan (amino acids having aromatic side chains);

lysine, arginine and histidine (amino acids having basic side chains);

aspartate and glutamate (amino acids with acidic side chains);

asparagine and glutamine (amino acids having amide side chains); And,

cysteine and methionine (amino acids with sulfur-containing side chains).

Additional candidates for replacement are human acceptor framework amino acids that are unusual or “rare” for human immunoglobulin at that position. Such amino acids may be replaced by amino acids from the equivalent position of the donor antibody or from equivalent positions of more typical human immunoglobulins. For example, a substitution may be desirable if an amino acid in the human acceptor immunoglobulin framework is uncommon at that position, but the corresponding amino acid in the donor immunoglobulin is common at that position in human immunoglobulin sequences; or if the amino acid in the acceptor immunoglobulin is rare at that position and the corresponding amino acid in the donor immunoglobulin is also rare relative to other human immunoglobulin sequences. These criteria help ensure that an atypical amino acid in the human framework region will not disrupt the structure of the antibody. Moreover, by replacing the unusual amino acid of the human acceptor sequence with an amino acid from the donor antibody that is typical of human antibodies, the humanized antibody can be made less immunogenic.

As used herein, the term “rare” means an amino acid that occupies a given position in less than about 20%, typically less than about 10%, of the sequences in a representative sequence sample, and the term “common” as used herein means an amino acid that occupies a given position in more than about 25%, typically more than about 50% of the sequences in a representative sample. For example, all human light and heavy chain variable region sequences are respectively grouped into “subgroups” of sequences that are particularly homologous to each other and share the same amino acids at certain important positions (Kabat et al., supra). When deciding whether an amino acid in a human acceptor sequence is “rare” or “common” among human sequences, it is often preferable to consider only human sequences that are in the same subgroup as the acceptor sequence.

Additional candidates for replacement are acceptor scaffold residues that correspond to rare or unusual donor scaffold residues. Rare or unusual donor scaffold residues are residues that are rare or unusual (as defined herein) for donor antibodies at a given position. For donor antibodies, a subgroup can be defined according to the Kabat system and residue positions that differ from the consensus can be identified. These specific differences in the donor sequence may indicate somatic mutations in the donor sequence that result in increased activity. Unusual residues predicted to affect binding are retained, while residues predicted to be unimportant for binding may be replaced.

Additional candidates for replacement are non-germline residues present in the acceptor framework region. For example, when aligning an antibody acceptor chain (i.e., a human antibody chain that has significant sequence identity with the donor antibody chain) with a germline antibody chain (likewise having significant sequence identity with the donor chain), residues that do not match the backbone of the acceptor chain and germline chain framework may be replaced by corresponding residues from the germline sequence.

With the exception of the specific amino acid substitutions described above, the framework regions of humanized immunoglobulins are generally substantially identical, and more often, identical to the framework regions of the human antibodies from which they are derived (except as described herein for the purposes of the present invention). Of course, many of the amino acids in the framework region make little or no direct contribution to the specificity or affinity of the antibody. Therefore, many individual conservative substitutions of framework residues can be made without noticeable changes in the specificity or affinity of the resulting humanized immunoglobulin. Thus, in one embodiment, the humanized immunoglobulin variable region framework has at least 65, 75, or 85% sequence similarity, or identity, to a human variable region framework sequence or a consensus of such sequences. In another embodiment, the humanized immunoglobulin variable region framework has at least 90%, preferably 95%, more preferably 96%, 97%, 98%, or 99% sequence similarity, or identity, to a human variable region framework sequence or a consensus of such sequences. However, such substitutions are generally not advisable (except as described herein).

The degree of identity and similarity referred to herein can be readily calculated, for example, as described in Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing. Informatics and Genome Projects, Smith, D.W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A.M., and Griffin, H.G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987, Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991, BLAST™ program, available from NCBI (Altschul, S.F. et al., 1990, J. Mol. Biol. 215:403-410; Gish, W. & States, D.J. 1993, Nature Genet. 3:266-272. Madden, T.L. et al., 1996, Meth. Enzymol. 266:131-141; Altschul, S. F. et al., 1997, Nucleic Acids Res. 25: 3389-3402; Zhang, J. & Madden, T. L. 1997, Genome Res. 7: 649-656, all publications are incorporated herein by reference.

A number of reviews have been published discussing CDR-grafted antibodies, including a publication by Vaughan et al. (Nature Biotechnology, 16, 535-539, 1998), the contents of which are incorporated herein by reference.

Anti-IgE antibodies of the present invention may also contain additional binding domains, for example, like the DVD-Ig molecule, which is disclosed in WO2007/024715, or the so-called antibody (FabFv) ₂ Fc, described in WO2011/030107. Thus, the antibody used in the present invention includes bi-, tri- or tetravalent full-length antibodies.

Antigen binding fragments

Antigen-binding fragments include single-chain antibodies (ie, full-length heavy chain and light chain); Fab, modified Fab, Fab', modified Fab', F(ab') ₂ , Fv, Fab-Fv, Fab-dsFv, single domain antibodies (eg VH or VL or VHH), for example those described in WO2001090190, scFv, bi -, tri- or tetravalent antibodies, bis-scFv, diabodies, tribodies, tribodies, tetrabodies, and antigen epitope-binding fragments of any of the above (see, for example, Holliger and Hudson, 2005, Nature Biotech. 23(9):1126-1136; Adair and Lawson, 2005, Drug Design Reviews - Online 2(3), 209-217). Methods for creating and producing such antibody fragments are well known in the art (see, for example, Verma et al., 1998, Journal of Immunological Methods, 216, 165-181). The Fab-Fv format was first described in WO2009/040562, and its disulfide bond-stabilized variants, Fab-dsFv, were first described in WO2010/035012. Other antibody fragments for use in the present invention include the Fab and Fab' fragments described in international patent applications WO2005/003169, WO2005/003170 and WO2005/003171. Multivalent antibodies may have multiple specificities, for example, be bispecific, or may be monospecific (see, for example, WO92/22583 and WO05/113605). One example of the latter is tri-Fab (or TFM) described in WO92/22583.

A typical Fab' molecule contains a pair of heavy and light chains, with the heavy chain comprising a VH variable region, a CH1 constant domain, and a native or modified hinge region, and the light chain comprising a VL variable region and a CL constant domain.

In one embodiment, a Fab' dimer of the present invention is provided to create F(ab') ₂ , for example, the dimerization can be through a natural hinge sequence described herein or a derivative thereof, or a synthetic hinge sequence.

An antibody binding domain will typically contain 6 CDR regions, three from the heavy chain and three from the light chain. In one embodiment, the CDR regions together with the framework form a variable region. Thus, in one embodiment, the antigen binding fragment comprises an IgE-specific binding domain comprising a light chain variable region and a heavy chain variable region.

It should be understood that one or more (e.g., 1, 2, 3 or 4) amino acid substitutions, additions and/or deletions may be introduced into the CDR regions or other sequences (e.g., variable domains) provided by the present invention, as described above or lower, without significantly changing the ability of the antibody to bind to IgE. The effect of any of the amino acid substitutions, additions and/or deletions can be readily tested by one skilled in the art, for example, using the methods described herein, particularly in the Examples section.

In one embodiment, one or more (e.g., 1, 2, 3, or 4) amino acid substitutions, additions, and/or deletions may be introduced into the CDR regions or framework regions used in the antibody or fragment of the present invention, thus that the binding affinity ( _KD ) of the anti-IgE antibody, or antigen-binding fragment, of the invention to IgE is less than 2, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1 .3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4 or 0.3 nM. In one embodiment, a modified humanized antibody is provided in which modifications have been made to either the CDR regions, framework regions, or both, to reduce the _KD , for example, to less than 2, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, 0, 5, 0.4 or 0.3 nM.

The antibody fragment of the present invention includes Fab, Fab′, F(ab′) ₂ , scFv, diabody, scFAb, dFv, single-domain antibody light chain, dsFv, CDR-containing peptide, and the like.

Fab is an antibody fragment having a molecular weight of approximately 50,000 and antigen-binding activity, in which approximately half of the N-terminal portion of the H chain and the entire L chain are among the fragments obtained by treating IgG with the protease papain (cleaving at the amino acid residue at position 224 H chains) are linked together by a disulfide bond.

The Fab of the present invention can be obtained by treating a human CDR-grafted antibody of the present invention that specifically reacts with the IgE protease papain. In addition, Fab can be produced by inserting DNA encoding Fab antibodies into a prokaryotic cell expression vector or eukaryotic cell expression vector and introducing the vector into prokaryotic or eukaryotic cells to express the Fab.

F(ab′) ₂ is an antibody fragment having a molecular weight of approximately 100,000 and antigen binding activity slightly larger than Fab fragments linked by a disulfide bond at the hinge region, and is among the fragments produced by treatment of IgG protease with pepsin.

The F(ab′) ₂ of the present invention can be produced by treating a human CDR-grafted antibody that specifically reacts with the IgE protease with pepsin. In addition, F(ab′) ₂ can be obtained by linking the Fab′ described below with a thioether bond or a disulfide bond.

Fab′ is an antibody fragment having a molecular weight of approximately 50,000 and antigen-binding activity that is produced by cleavage of the disulfide bond at the F(ab′) ₂ hinge region.

Fab' of the present invention can be obtained by treating F(ab') ₂ specifically reacting with IgE, the reducing reagent, dithiothreitol. In addition, the Fab′ of the present invention can be obtained by inserting DNA encoding the Fab′ of a human CDR-grafted antibody of the present invention specifically reactive with IgE into a prokaryotic cell expression vector or eukaryotic cell expression vector and introducing the vector into prokaryotic or eukaryotic cells. cells for Fab′ expression.

scFv is a VH-P-VL or VL-P-VH polypeptide in which one VH chain and one VL chain are linked using an appropriate peptide linker (P) of 12 or more residues, and which has antigen-binding activity.

The scFv of the present invention can be obtained by obtaining cDNAs encoding the VH and VL of the human CDR-grafted antibody specifically reactive with IgE of the present invention, constructing the DNA encoding the scFv, inserting the DNA into a prokaryotic cell expression vector or an eukaryotic cell expression vector, and subsequent introduction of the expression vector into prokaryotic or eukaryotic cells to express the scFv.

The Fab fragment of the present invention can be linked, directly or via a linker, to scFv. As used herein, the term “single chain variable fragment,” or “scFv,” means a single chain variable fragment that is stabilized by a peptide linker between the VH and VL variable domains, for example, a peptide linker with the amino acid sequence of SEQ ID NO:151. The connection to the Fab fragment can be formed by chemical conjugation, but the most preferred is a translational fusion, that is, a genetic fusion product with the sequence of each of the components encoded in the expression vector. Thus, the linker is typically an amino acid linker as described herein. The scFv of the present invention associated with a Fab fragment can bind to a serum carrier protein to increase the half-life of the antibody fusion protein in vivo. In this case, the increase in half-life is independent of IgE binding and may be beneficial.

As used herein, the term “serum carrier protein” means any suitable carrier protein in plasma to which the scFv can bind, in one example the serum carrier protein is selected from thyroxine binding protein, transthyretin, α1-acid glycoprotein, transferrin, fibrinogen and albumin , or a fragment of any of them. Typically, scFv binds to albumin, preferably human serum albumin.

Any suitable albumin-binding scFv can be included in the antibody fusion proteins of the invention. Suitable albumin-binding domains are known in the art.

A diabody is an antibody fragment in which scFv fragments having the same or different antigen binding specificities form a dimer, and which has bivalent antigen binding activity for the same antigen, or two specific antigen binding activities for different antigens.

The diabody of the present invention, for example, a divalent diabody that specifically reacts with IgE, can be prepared by obtaining cDNAs encoding VH and VL antibodies that specifically react with IgE, constructing DNA encoding a scFv fragment having a polypeptide linker of 3-10 residues, inserting the DNA into an expression vector for prokaryotic cells or an expression vector for eukaryotic cells and then introducing the vector into prokaryotic or eukaryotic cells to express the diabody.

dsFv is produced by linking polypeptides in which one amino acid residue of each of VH and VL is replaced by a cysteine residue, a disulfide bond between cysteine residues. The amino acid residue that is replaced with a cysteine residue can be selected based on an assessment of the three-dimensional structure of the antibody by the method described by Reiter et al. (Protein Engineering, 7, 697 (1994)).

The dsFv of the present invention can be produced by obtaining cDNAs encoding the VH and VL of the human CDR-grafted antibody specifically reactive with IgE of the present invention, constructing the DNA encoding the dsFv, inserting the DNA into a prokaryotic cell expression vector or an eukaryotic cell expression vector, and subsequent introduction of the vector into prokaryotic or eukaryotic cells to express dsFv.

A CDR-containing peptide is constructed by including at least one of the H chain and L chain CDR regions. Several CDR regions can be linked directly or through a corresponding peptide linker.

The CDR-containing peptide of the present invention can be obtained by obtaining cDNA encoding the CDRs of the VH and VL of a human CDR-grafted antibody specifically reactive with IgE, constructing the DNA encoding the CDR, inserting the DNA into a prokaryotic cell expression vector or an eukaryotic cell expression vector and subsequent introduction of the vector into prokaryotic or eukaryotic cells for expression of the peptide. In addition, the CDR-containing peptide can also be produced by a chemical synthesis method such as the Fmoc method (fluorenyl methoxycarbonyl method), the tBoc method (t-butyloxycarbonyl method) or the like.

The antibody of the present invention includes antibody derivatives in which a radioisotope, protein, agent or the like is chemically or genetically conjugated to the antibody of the present invention.

The antibody derivatives of the present invention can be prepared by chemically conjugating a radioisotope, protein or agent to the N-terminal portion or C-terminal portion of the H chain or L chain of an antibody, or an antibody fragment specifically reactive with IgE, with an appropriate substituent group or side chain an antibody or antibody fragment, or with a sugar chain in the antibody or antibody fragment (Antibody Engineering Handbook, edited by Osamu Kanemitsu, published by Chijin Shokan (1994)).

In addition, they can be obtained by genetic engineering methods by linking DNA encoding an antibody, or antibody fragment, of the present invention that specifically reacts with IgE, with another DNA encoding the protein to be bound, inserting the DNA into an expression vector, and introducing an expression vector into the host cell.

The radioisotope includes ¹³¹ I, ¹²⁵ I and the like, and it can be conjugated to the antibody, for example, by the chloramine T method.

The agent is preferably a low molecular weight compound. Examples include anticancer agents such as alkylating agents (eg, nitrogen mustard, cyclophosphamide), metabolic antagonists (eg, 5-fluorouracil, methotrexate), antibiotics (eg, daunomycin, bleomycin, mitomycin C, daunorubicin, doxorubicin), plant alkaloids (eg , vincristine, vinblastine, vindesine), hormonal drugs (eg, tamoxifen, dexamethasone) and the like (Clinical Oncology, edited by the Japanese Society of Clinical Oncology, published by Cancer and Chemotherapy (1996)); anti-inflammatory drugs such as steroids (eg, hydrocortisone, prednisone), non-steroidal drugs (eg, aspirin, indomethacin), immunomodulators (eg, aurothiomalate, penicillamine), immunosuppressants (eg, cyclophosphamide, azathioprine), and antihistamines (eg, chlorpheniramine maleate, clemastine) (Inflammation and Anti-inflammatory Therapy, Ishiyaku Shuppan (1982)); etc. Methods for conjugating daunomycin with an antibody include a method in which daunomycin and the amino group of an antibody are conjugated using glutaraldehyde, a method in which the amino group of daunomycin and a carboxyl group of an antibody are conjugated using a water-soluble carbodiimide, and the like.

In addition, a toxin such as ricin, diphtheria toxin and the like can be used to directly inhibit cancer cells. For example, an antibody-protein fusion product can be produced by linking a cDNA encoding an antibody or antibody fragment to another cDNA encoding a protein, constructing the DNA encoding the fusion antibody, inserting the DNA into a prokaryotic cell expression vector or eukaryotic cell expression vector, and then introducing the vector into prokaryotic or eukaryotic cells to express the fusion antibody.

The present invention also provides antibody fragments or antigen binding fragments, including fusion products of binding fragments, for example, immunoglobulin-like fragments and compounds, such as diabodies, scAbs, bispecific fragments, tribodies, Fab-Fv-Fv, Fab-Fv, tribodies, ( Fab-Fv) ₂ -Fc, as well as fragments or portions of antibodies, such as CDR regions or antibody loops containing CDR regions, which are grafted onto non-Ig scaffolds, such as fibronectin or leucine zippers, as described in Binz et al ., (2005) Nat. Biotech. 23:1257-1268, the contents of the publication are included in this document in full.

Anti-IgE conjugated monoclonal antibodies and antigen binding fragments

If desired, an antibody, or antigen binding fragment, for use in the present invention can be conjugated to one or more effector molecules. It should be understood that the effector molecule can be one effector molecule, or two or more such molecules linked into a single fragment that can be attached to the antibodies of the present invention. If an antibody fragment linked to an effector molecule is desired, it can be produced by standard chemical methods or by recombinant DNA techniques in which the antibody fragment is linked either directly or via a cross-linker to the effector molecule. Methods for conjugating such effector molecules to antibodies are well known in the art (see Hellstrom et al., Controlled Drug Delivery, 2nd edition, Robinson et al., eds., 1987, pp. 623-53; Thorpe et al., 1982, Immunol. Rev., 62:119-58 and Dubowchik et al., 1999, Pharmacology and Therapeutics, 83, 67-123). Specific chemical methods include, for example, those described in WO93/06231, WO92/22583, WO89/00195, WO89/01476 and WO03/031581. Alternatively, if the effector molecule is a protein or polypeptide, coupling can be accomplished by recombinant DNA techniques, for example as described in WO86/01533 and EP0392745.

As used herein, the term "effector molecule" includes, for example, antineoplastic agents, drugs, toxins, biologically active proteins such as enzymes, other antibodies or antibody fragments, antigen binding fragments, synthetic (including PEG) or natural polymers, nucleic acids and fragments thereof, such as DNA, RNA and their fragments, radionuclides, in particular radioactive iodine, radioactive isotopes, chelated metals, nanoparticles and reporter groups such as fluorescent compounds or compounds that can be detected by NMR or ESR spectroscopy.

Examples of effector molecules may include cytotoxins or cytotoxic agents, including any agent that harms cells (eg, kills). Examples include combrestatins, dolastatins, epothilones, staurosporine, maytansinoids, spongistatins, rhizoxin, halichondrins, roridins, hemiasterlins, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, downora bicine , dihydroxyanthracinedione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol and puromycin, as well as their analogs or homologues.

Effector molecules also include, but are not limited to, antimetabolites (eg, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, decarbazine), alkylating agents (eg, mechlorethamine, thiotepa, chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibrommannite, streptozotocin, mitomycin C and cis-dichlorodiamine platinum(II) (DDP, cisplatin), anthracyclines (eg, daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (eg, dactinomycin ( formerly actinomycin), bleomycin, mithramycin, anthramycin (AMC), calicheamicins or duocarmycins), and antimitotic agents (eg, vincristine and vinblastine).

Other effector molecules may include chelated radionuclides such as ¹¹¹ In and ⁹⁰ Y, Lu ¹⁷⁷ , bismuth ²¹³ , californium ²⁵² , iridium ¹⁹² and tungsten ¹⁸⁸ /rhenium ¹⁸⁸ ; or drugs such as, but not limited to, alkylphosphocholines, topoisomerase I inhibitors, taxoids and suramin.

Other effector molecules include proteins, peptides and enzymes. Enzymes of interest include, but are not limited to, proteolytic enzymes, hydrolases, lyases, isomerases, transferases. Proteins, polypeptides and peptides of interest include, but are not limited to, immunoglobulins, toxins such as abrin, ricin A, pseudomonas exotoxin or diphtheria toxin, proteins such as insulin, tumor necrosis factor, α-interferon, β-interferon, factor nerve growth factor, platelet-derived growth factor or tissue plasminogen activator, antithrombotic agents or antiangiogenic agents, for example angiostatin or endostatin, or biological response modifiers such as lymphokine, interleukin-1 (IL-1), interleukin-2 (IL-2), granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), nerve growth factor (NGF) or other growth factors and immunoglobulins.

Other effector molecules may include detectable substances useful, for example, in diagnostics. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radionuclides, positron active metals (for use in positron emission tomography) and non-radioactive paramagnetic metal ions. See generally US Pat. No. 4,741,900 for information on metal ions that can be conjugated to antibodies for diagnostic use. Suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase or acetylcholinesterase; suitable prosthetic groups include streptavidin, avidin and biotin; suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride and phycoerythrin; Suitable luminescent materials include luminol; suitable bioluminescent materials include luciferase, luciferin and aequorin; and suitable radionuclides include ¹²⁵ I, ¹³¹ I, ¹¹¹ In and ⁹⁹ Tc.

In another example, an effector molecule may increase the half-life of an antibody in vivo and/or decrease the immunogenicity of the antibody, and/or facilitate delivery of the antibody across the epithelial barrier to the immune system. Examples of suitable effector molecules of this type include polymers, albumin, albumin-binding proteins or albumin-binding compounds such as those described in WO05/117984.

In one embodiment, the half-life that the effector molecule provides independent of the IgE or anti-IgE human antibody is advantageous.

If the effector molecule is a polymer, it will typically be a synthetic or natural polymer, such as an optionally substituted straight or branched chain polyalkylene, polyalkenylene or polyoxyalkylene polymer, or a branched or unbranched polysaccharide, such as a homo- or heteropolysaccharide.

Specific optional substituents that may be present on the above synthetic polymers include one or more hydroxy, methyl or methoxy groups.

Specific examples of synthetic polymers include optionally substituted poly(ethylene glycol), poly(propylene glycol), straight or branched chain poly(vinyl alcohol), or derivatives thereof, particularly optionally substituted poly(ethylene glycol) such as methoxy poly(ethylene glycol) or the same. derivatives.

Specific natural polymers include lactose, amylose, dextran, glycogen, or derivatives thereof.

In one embodiment, the polymer is albumin or a fragment thereof, such as human serum albumin or a fragment thereof. In one embodiment, the polymer is a PEG molecule.

As used herein, the term “derivatives” as used with reference to a conjugate includes reactive derivatives, for example, thiol-selective reactive groups such as maleimides, and the like. The reactive group can be linked to the polymer directly or through a linker segment. It should be understood that the remainder of such a group will in some cases form part of the product as a linking group between the antibody moiety and the polymer.

The size of the natural or synthetic polymer may vary as needed, but will generally be in the average molecular weight range of 500 Da to 50,000 Da, eg 5000-40,000 Da, eg 20,000-40,000 Da. The specific polymer size can be selected based on the intended use of the product, for example, localization to certain tissues such as tumors, or long half-life in the circulation (for review, see Chapman, 2002, Advanced Drug Delivery Reviews, 54, 531 -545). Thus, for example, if the product is to leave the circulation and enter tissue, for example for use in the treatment of a tumor, it may be preferable to use a low molecular weight polymer, for example with a molecular weight of about 5000 Da. For applications where the product remains in the circulation system, it may be preferable to use a higher molecular weight polymer, for example, having a molecular weight in the range of 20,000 Da to 40,000 Da.

Suitable polymers include a polyalkylene polymer such as poly(ethylene glycol) or, in particular, methoxypoly(ethylene glycol) or a derivative thereof, and especially with a molecular weight in the range of about 15,000 Da to about 40,000 Da.

In one example, antibodies for use in the present invention are linked to poly(ethylene glycol) (PEG) molecules. In one specific example, the antibody is an antibody fragment and the PEG molecules can be attached via any available amino acid side chain or terminal amino acid functional group found on the antibody fragment, such as any free amino, imino, thiol, hydroxyl or carboxyl group. Such amino acids may exist naturally in the antibody fragment or may be artificially introduced into the fragment using recombinant DNA techniques (see, for example, US5219996; US5667425; WO98/25971, WO2008/038024). In one example, the antibody molecule of the present invention is a modified Fab fragment in which the modification is the addition of one or more amino acids at the C-terminus of its heavy chain to allow attachment of an effector molecule. Preferably, the additional amino acids form a modified hinge region containing one or more cysteine residues to which the effector molecule can bind. Multiple sites can be used to attach two or more PEG molecules.

Preferably, the PEG molecules are covalently linked through the thiol group of at least one cysteine residue located in the antibody fragment. Each polymer molecule attached to a modified antibody fragment may be covalently linked to a sulfur atom of a cysteine residue located in the fragment. The covalent bond will typically be a disulfide bond or, in particular, a carbon disulfide bond. If a thiol group is used as an attachment point for suitably activated effector molecules, for example, thiol-selective derivatives such as maleimides and cysteine derivatives can be used. The activated polymer can be used as a starting material to produce polymer-modified antibody fragments as described above.

The activated polymer can be any polymer containing a thiol reactive group such as an α-halocarboxylic acid or an ester, eg iodoacetamide, an imide, eg maleimide, vinyl sulfone or disulfide. Such starting materials can be purchased (eg, from Nektar, formerly Shearwater Polymers Inc., Huntsville, AL, USA) or can be prepared from commercially available starting materials by conventional chemical methods. Specific PEG molecules include 20K methoxy-PEG-amine (available from Nektar, formerly Shearwater; Rapp Polymere and SunBio) and M-PEG-SPA (available from Nektar, formerly Shearwater).

In one embodiment, the antibody is a modified Fab fragment, Fab' fragment or diFab, which is PEGylated, that is, contains PEG (poly(ethylene glycol)) covalently attached to it, for example, by the method described in EP 0948544 or EP 1090037 [see also Poly(ethyleneglycol) Chemistry, Biotechnical and Biomedical Applications, 1992, J. Milton Harris (ed), Plenum Press, New York, Poly(ethyleneglycol) Chemistry and Biological Applications, 1997, J. Milton Harris and S. Zalipsky (eds), American Chemical Society, Washington DC and Bioconjugation Protein Coupling Techniques for the Biomedical Sciences, 1998, M. Aslam and A. Dent, Grove Publishers, New York; Chapman, A. 2002, Advanced Drug Delivery Reviews 2002, 54:531-545]. In one example, the PEG is attached to a cysteine residue in the hinge region. In one example, a PEG-modified Fab moiety has a maleimide group covalently linked to one thiol group in the modified hinge region. The lysine moiety may be covalently linked to a maleimide group, and a methoxypoly(ethylene glycol) polymer having a molecular weight of about 20,000 Da can be attached to each of the amino groups on the lysine moiety. The total molecular weight of the PEG attached to the Fab fragment may thus be approximately 40,000 Daltons.

Specific lysine-modifying PEG molecules include 2-[3-(N-maleimido)propionamido]ethylamide N,N'-bis(methoxypoly(ethylene glycol), MW 20000), otherwise known as PEG2MAL40K (available from Nektar, formerly Shearwater).

Alternative sources of PEG linkers include NOF, which provides GL2-400MA3 (where m in the structure below is 5) and GL2-400MA (where m is 2), and n is approximately 450:

That is, each PEG molecule has a molecular weight of approximately 20,000 Da.

Thus, in one embodiment, the PEG is 2,3-bis(methylpolyoxyethyleneoxy)-1-{[3-(6-maleimido-1-oxohexyl)amino]propyloxy}hexane (branched 2-arm PEG, -CH2) 3NHCO(CH2)5-MAL, MM 40000, known as SUNBRIGHT GL2-400MA3.

Other alternative PEG effector molecules have the following formula:

available from Dr Reddy, NOF and Jenkem.

In one embodiment, an antibody of the invention is provided that is PEGylated (e.g., using PEGs described herein) by attaching the PEG to a cysteine amino acid residue at or near position 226 in the chain, e.g., at position 226 of the heavy chains (with sequential numbering).

In one embodiment, the present invention provides a Fab'-PEG molecule comprising one or more PEG polymers, for example 1 or 2 polymers, such as a 40 kDa polymer or polymers.

The Fab'-PEG molecules of the present invention may have the particular advantage that they have a half-life independent of the Fc moiety. In one example, the present invention provides a method of treating a disease that is ameliorated by modulating the biological activity of human IgE, comprising administering a therapeutically effective amount of an anti-IgE antibody, or antigen binding fragment thereof, wherein the antibody, or antigen binding fragment thereof, has a half-life that is not depends on the binding of Fc to IgE.

In one embodiment, a Fab' fragment is provided conjugated to a polymer, such as a PEG molecule, a starch molecule, or an albumin molecule.

In one embodiment, a scFv fragment is provided conjugated to a polymer, such as a PEG molecule, a starch molecule, or an albumin molecule.

In one embodiment, the antibody, or fragment, is conjugated to a starch molecule, for example, to increase half-life. Methods for conjugating starch with protein are described in US8017739, the contents of which are incorporated herein by reference.

Polynucleotides

The present invention also relates to an isolated DNA sequence encoding the heavy and/or light chain(s) of the antibody molecule of the present invention. Preferably, the DNA sequence encodes the heavy or light chain of the antibody molecule of the present invention. The DNA sequence of the present invention may include synthetic DNA, such as chemically produced DNA, cDNA, genomic DNA, or any combination thereof.

DNA sequences encoding the antibody molecule of the present invention can be obtained by methods well known to those skilled in the art. For example, DNA sequences encoding part or all of the heavy and light chains of an antibody can be synthesized, if necessary, based on certain DNA sequences or based on corresponding amino acid sequences.

DNA encoding acceptor framework sequences is widely available to those skilled in the art and can be readily synthesized based on the corresponding known amino acid sequences.

Standard molecular biology techniques can be used to obtain DNA sequences encoding the antibody molecule of the present invention. The desired DNA sequences can be synthesized in whole or in part using oligonucleotide synthesis methods. Where appropriate, site-directed mutagenesis and polymerase chain reaction (PCR) methods can be used.

The present invention also relates to a cloning or expression vector containing one or more DNA sequences of the present invention. Accordingly, a cloning or expression vector is provided containing one or more DNA sequences encoding an antibody of the present invention. Preferably, the cloning or expression vector contains two DNA sequences encoding the light chain and the heavy chain of the antibody molecule of the present invention, respectively, and the corresponding signal sequences. In one example, the vector contains intergenic sequence between the heavy and light chain sequences (see WO03/048208).

General vector construction methods, transfection methods, and culture methods are well known to those skilled in the art. In this regard, reference is made to Current Protocols in Molecular Biology, 1999, F. M. Ausubel (ed), Wiley Interscience, New York and the Maniatis manual, published by Cold Spring Harbor Publishing.

Host cells expressing anti-IgE antibodies or fragments thereof

Also provided is a host cell containing one or more cloning, or expression, vectors containing one or more DNA sequences encoding an antibody of the present invention. Any suitable host cell/vector system can be used to express DNA sequences encoding the antibody molecule of the present invention. Bacterial, eg E. coli, and other microbial systems can be used, or eukaryotic expression systems can also be used, eg with mammalian host cells. Suitable mammalian host cells include CHO, myeloma or hybridoma cells.

Suitable types of Chinese hamster ovary cells (CHO cells) for use in the present invention may include CHO and CHO-K1 cells, including dhfr-CHO cells such as CHO-DG44 cells and CHO-DXB11 cells, which can be used with selective DHFR marker, or CHOK1-SV cells, which can be used with the glutamine synthetase selectable marker. Other types of cells used for antibody expression include lymphocytic cell lines, for example, NSO myeloma cells and SP2 cells, COS cells. Other suitable cells may include human embryonic kidney (hek) fibroblasts, for example hek293F and ExpiHek cells, which are known in the art.

CHO cells are preferred for full-length Abs of the invention because they are the standard host cells for producing omalizumab (in one embodiment, providing the antibodies of the invention with a standard glycosylation pattern of omalizumab) [see also WO2013/181577].

Preparation of anti-IgE antibodies or their fragments

The present invention also provides a method for producing an antibody molecule of the present invention, comprising culturing a host cell containing a vector of the present invention under conditions suitable to initiate expression of a protein with DNA encoding the antibody molecule of the present invention, and isolating the antibody molecule.

The antibody molecule may contain only a heavy or light chain polypeptide, in which case only the sequence encoding the heavy chain or light chain polypeptide must be used to transfect host cells. To produce products containing both heavy and light chains, a cell line can be transfected with two vectors, a first vector encoding a light chain polypeptide and a second vector encoding a heavy chain polypeptide. Alternatively, a single vector containing sequences encoding light chain and heavy chain polypeptides can be used.

A method is provided for culturing a host cell and expressing an antibody or fragment thereof, isolating the latter and, optionally, purifying it to obtain the isolated antibody or fragment. In one embodiment, the method further includes the step of conjugating the effector molecule to the isolated antibody or fragment, for example, conjugation to a PEG polymer, particularly as described herein.

In one embodiment, a method for purifying an antibody (specifically, an antibody or fragment of the invention) is provided, comprising the steps of: performing anion exchange chromatography in a non-binding mode such that impurities are retained on the column and the antibody is eluted.

In one embodiment, purification involves affinity retention on a Protein A column, followed by titration. In one embodiment, purification involves affinity retention on a protein G column, followed by HPLC titration. In one embodiment, purification involves affinity retention on an IgE column, followed by titration.

In one embodiment, purification includes the use of Cibacron blue or a similar reagent to purify albumin fusion or conjugated molecules.

Suitable ion exchange resins for use in the method include Q.FF resin (available from GE-Healthcare). This step can, for example, be carried out at a pH value of approximately 8.

The method may further include an initial capture step using cation exchange chromatography, performed, for example, at a pH value of about 4-5, for example 4.5. For cation exchange chromatography, a resin such as CaptoS or SP Sepharose FF (available from GE-Healthcare) can, for example, be used. The antibody or fragment can then be eluted from the resin using an ionic saline solution such as sodium chloride, for example, at a concentration of 200 mM.

Thus, the chromatography step or steps may include one or more washing steps, depending on the situation.

The purification method may also include one or more filtration steps, such as a diafiltration step or an HPLC filtration step.

Thus, in one embodiment, a purified anti-IgE antibody or fragment is provided, for example a humanized antibody or fragment, in particular an antibody or fragment according to the invention, in a substantially purified form, in particular free or substantially free of endotoxin and/or protein or DNA of the host cell.

As used above, the term "purified form" should mean a degree of purity of at least 90%, for example, 91, 92, 93, 94, 95, 96, 97, 98, 99% by weight content, or an even greater degree of purity.

“Substantially free of endotoxin” generally means an endotoxin content of 1 IU per mg of antibody product or less, such as 0.5 or 0.1 IU per mg of product.

“Substantially free of host cell protein or DNA” generally means a host cell protein and/or DNA content of 400 μg per mg of antibody product or less, such as 100 μg per mg or less, e.g., 20 mcg per mg, depending on the situation.

Pharmaceutical compositions

Because the antibodies of the present invention are useful for the treatment and/or prevention of a pathological condition, the present invention also provides a pharmaceutical or diagnostic composition containing an antibody, or antigen binding fragment, of the present invention in combination with one or more of a pharmaceutically acceptable excipient, diluent or carrier. Accordingly, the use of an antibody, or antigen-binding fragment, of the invention for the production of a medicinal product is provided. The composition will typically be provided as part of a sterile pharmaceutical composition, which typically contains a pharmaceutically acceptable carrier. In addition, the pharmaceutical composition of the present invention may contain a pharmaceutically acceptable excipient.

The present invention also relates to a method for preparing a pharmaceutical or diagnostic composition, comprising adding and mixing an antibody, or antigen binding fragment, of the present invention with one or more of a pharmaceutically acceptable excipient, diluent or carrier.

The antibody, or antigen binding moiety, may be the sole active ingredient in a pharmaceutical or diagnostic composition, or may be accompanied by other active ingredients, including other antibodies or non-antibody ingredients, such as steroid or other drug molecules, in particular drug molecules, whose half-life is independent of IgE binding.

Preferably, the pharmaceutical compositions contain a therapeutically effective amount of an antibody, or antigen binding fragment, of the invention. As used herein, the term “therapeutically effective amount” means the amount of a therapeutic agent necessary to treat, ameliorate, or prevent the target disease or condition, or to achieve a noticeable therapeutic or preventive effect. For any disclosed antibody or antigen binding fragment, the therapeutically effective amount can be assessed initially either in cell culture assays or in animal models, typically rodents, rabbits, dogs, pigs or primates. An animal model can also be used to determine the appropriate concentration range and route of administration. Such information can be used to determine human doses and routes of administration.

The exact therapeutically effective amount for a human individual will depend on the severity of the disease state, the general health of the individual, the age, body weight and sex of the individual, diet, timing and frequency of administration, drug combination(s), sensitivity of response and resistance/response to therapy. This amount can be determined by normal experimentation and is within the competence of the attending physician. Typically, a therapeutically effective amount will be from 0.01 mg/kg to 500 mg/kg, such as 0.1 mg/kg to 200 mg/kg, such as 100 mg/kg. Pharmaceutical compositions may preferably be provided in unit dosage forms containing a predetermined amount of the active agent of the invention in each dose.

Therapeutic doses of antibodies, or antigen-binding fragments, of the present invention do not cause obvious toxicological effects in vivo.

Preferably, IgE activity in vivo can be maintained at a suitably reduced level by sequential dosing of the antibody, or binding fragment, of the invention.

The compositions may be administered to a patient alone or may be administered in combination (eg, simultaneously, sequentially, or separately) with other agents, drugs, or hormones.

The pharmaceutical composition may also contain a pharmaceutically acceptable carrier for administering the antibody or antigen binding fragment. The carrier itself should not induce the production of antibodies harmful to the individual receiving the composition and should not be toxic. Suitable carriers may be large, slowly metabolized macromolecules such as proteins, polypeptides, liposomes, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, copolymers of amino acids and inactive viral particles.

Pharmaceutically acceptable salts may be used, for example salts of mineral acids such as hydrochlorides, hydrobromides, phosphates and sulfates, or salts of organic acids such as acetates, propionates, malonates and benzoates.

In addition, pharmaceutically acceptable carriers in therapeutic compositions may contain liquids such as water, saline, glycerin and ethanol. In addition, auxiliary substances such as wetting agents or emulsifiers or pH adjusters may be present in such compositions. Such carriers allow pharmaceutical compositions to be formulated into tablets, pills, dragees, capsules, liquids, gels, syrups, thick suspensions and suspensions for oral administration by the patient.

Preferred administration forms include those suitable for parenteral administration, for example by injection or infusion, for example by bolus injection or continuous infusion. If the preparation is intended for injection or infusion, it may be in the form of a suspension, solution or emulsion in an oily or aqueous medium, and it may contain auxiliary substances such as suspending agents, preservatives, stabilizers and/or dispersing agents. Alternatively, the antibody molecule may be in a dry form intended to be reconstituted in a suitable sterile liquid prior to use.

Once formulated, the compositions of the invention can be administered directly to the individual. The individuals receiving treatment may be animals. However, it is preferable if the compositions are adapted for administration to human subjects.

The pharmaceutical compositions of this invention can be administered by a variety of routes of administration, including, but not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, transdermal, transdermal (for example, see WO98/20734), subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, intravaginal or rectal routes. Hyposprays can also be used to administer the pharmaceutical compositions of the invention. Typically, therapeutic compositions can be prepared as injectable preparations, either as liquid solutions or suspensions. Solid forms suitable for dissolution or suspension in liquid media prior to injection may also be prepared.

Direct delivery of the compositions will generally be accomplished by subcutaneous (in particular), intraperitoneal, intravenous or intramuscular injection, or delivered into the interstitial space of the tissue. The compositions can also be injected into the site of injury. Doses may be administered in a single-dose system or a multiple-dose system.

It is understood that the active ingredient in the composition will be an antibody molecule. Because of this, it will be subject to degradation in the gastrointestinal tract. Thus, if the composition is to be administered through the gastrointestinal tract, the composition will need to contain agents that protect the antibody from degradation but release the antibody once it is absorbed from the gastrointestinal tract.

A detailed description of pharmaceutically acceptable carriers can be found in Remington's Pharmaceutical Sciences (Mack Publishing Company, N.J. 1991).

Structural and functional properties

In one aspect of the present invention, the antibody, or antigen binding fragment, binds to free and FcεRI-bound human IgE. When an antibody (or antigen binding fragment) of the invention binds to FcεRI-bound human IgE, it stabilizes the conformation of the IgE. In such a stabilized conformation, IgE has weaker binding affinity for FcεRI or omalizumab (or fragments thereof) than in the absence of the antibody or antigen binding fragment of the present invention, and FcεRI-bound human IgE dissociates from FcεRI. Preferably, the IgE, after dissociation from the FcεRI, remains associated with the antibody, or antigen binding fragment, described herein. As will be shown later herein (eg, in Example 1 and Figure 2), the antibody, or antigen binding fragment, of the invention binds to IgE in a conformation that is different from the conformation that IgE has when bound to omalizumab.

Without being bound by theory, the antibody of the present invention forces IgE to adopt a partially curved conformation (FIGS. 2C and 5C-D), thereby causing unbending structures characteristic of free or FcεRI-bound IgE. This extension prevents IgE from binding or maintaining binding to FcεRI. As will be shown in the Examples section, since the antibody of the present invention competes for the binding site on IgE with FcεRI, it is believed that the antibody may be capable of forming a complex with FcεRI-bound IgE by changing the structure of FcεRI-bound IgE, and stimulation of its dissociation from FcεRI. Antibodies of the present invention having these properties are those described herein, such as antibodies, or antigen binding fragments, containing:

a. a heavy chain variable region comprising SEQ ID NO:1, and a light chain variable region comprising:

i. SEQ ID NO:109; or

ii. SEQ ID NO:113; or

iii. SEQ ID NO:121; or

iv. SEQ ID NO:132; or

v. SEQ ID NO::139; or

b. SEQ ID NO:5 and

i. SEQ ID NO:24 in which S77 and S79 are replaced by Q;

ii. SEQ ID NO:117 or

iii. SEQ ID NO:125; or

iv. SEQ ID NO::136; or

v. SEQ ID NO:143.

Anti-IgE antibodies, or antigen-binding fragments having such properties, contact, or contact and are specific for, an epitope containing, with reference to SEQ ID NO:108, residues T373, W374, S375, R376, A377, S378, G379, P381 , Q417, C418, R419, P426, R427, A428 of the Cε3 domain and residues D278 and T281 of the human IgE Cε2 domain.

The antibodies of the present invention have methionine residues at certain positions. Oxidation of methionine residues is one of the most common pathways of protein degradation. Antibodies of the present invention in which methionine residues have been introduced at positions S64 and S71, with reference to SEQ ID NO:20, can be completely oxidized without affecting the ability of the antibodies to accelerate the dissociation of the IgE-Fc:sFcRIα complex.

Thus, the present invention also provides an anti-IgE antibody, or antigen binding fragment, which contains a heavy chain variable region having the amino acid sequence of SEQ ID NO:1, and a light chain variable region containing CDR-L2 and FW-L3, wherein the light chain variable region has the amino acid sequence of SEQ ID NO:20, except that CDR-L2 has the amino acid sequence of SEQ ID NO:50, and that FW-L3 has the amino acid sequence of SEQ ID NO:131 or 138, wherein methionine residues at positions 64 and/or 71, with reference to SEQ ID NO:20, are oxidized.

The present invention also provides an anti-IgE antibody, or antigen binding fragment, comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO:1, and a light chain variable region having the amino acid sequence of SEQ ID NO:132 or 139, wherein methionine residues at positions 64 and/or 71, with reference to SEQ ID NO:132 or 139, are oxidized.

Other methionine residues in the antibodies of the present invention can be oxidized without affecting the ability of the antibodies to accelerate the dissociation of the IgE-Fc:sFcRIα complex.

The invention will now be described by way of examples with reference to the embodiments illustrated in the accompanying drawings.

Examples

Example 1: Structure of an IgE-Fc-bound mutant of the therapeutic anti-IgE antibody omalizumab provides insight into its mechanism of action

annotation

Immunoglobulin E and its interaction with FcεRI and CD23 receptors play a central role in allergic diseases. Omalizumab, a clinically approved therapeutic antibody, inhibits the interaction between IgE and FcεRI, preventing mast cell and basophil activation, and blocks IgE binding to CD23. We solved the crystal structure of a 2:1 complex between omalizumab-derived Fab and IgE-Fc, with one Fab fragment associated with each Cε3 domain (but only one of the Fab fragments associated with the Cε2 domain). Although free IgE-Fc in solution is predominantly sharply curved, in the complex it is only partially curved, which prevents interaction with FcεRI; CD23 binding is sterically inhibited due to overlapping binding sites on each Cε3 domain. Solution state interaction analysis demonstrates the orthosteric and allosteric basis for inhibition of both receptor interactions and, together with the structure, reveals how omalizumab (and in particular the described omalizumab mutants) can accelerate the dissociation of receptor-bound IgE from FcεRI using intrinsic dynamics and allosteric potential of IgE.

Introduction

Immunoglobulin E (IgE) antibodies play an important role in allergic diseases by binding to allergens with their Fab arms and exerting their effector functions by binding to Fc region receptors ¹ . The two main IgE receptors are FcεRI and CD23/FcεRII, which are commonly referred to as high- and low-affinity receptors, respectively. On mast cells and basophils, IgE binds to FcεRI so tightly (K _D ≈ 10 ^-10 M ^-1 ) that such cells are sensitized by pre-bound IgE, and only the presence of an allergen is necessary to cross-link the IgE/FcεRI complexes and cause an immediate reaction. CD23 is a homotrimer and thus the initially low affinity of each IgE-binding C-type lectin-like head domain (K _D ≈ 10 ^-7 M ^-1 ) can be enhanced by the avidity effect when binding to aggregated IgE in immune complexes , and almost match the affinity of FcεRI for IgE ² . CD23, expressed on B cells, is involved in the regulation of IgE, and expressed on airway and intestinal epithelial cells, mediates transcytosis of IgE/allergen complexes ^1,2 . Both FcεRI and CD23 are expressed on different antigen presenting cells. Thus, IgE–receptor interactions are involved in several aspects of the allergic response, and IgE has been a long-standing target for therapeutic intervention ³ .

The Fc region of IgE includes a disulfide-linked dimer of three domains: Cε2, Cε3 and Cε4. Early FRET studies of chimeric IgE ^4,5 and X-ray scattering studies of IgE-Fc ⁶ solutions indicated a compact, curved structure, and later studies of the crystal structure of IgE-Fc revealed a sharply and asymmetrically curved conformation, with a pair of (Cε2) ₂ domains , folded back onto the Cε3 and Cε4 domains ⁷ . The bend, defined as the angle between the local dual axis of domain pair (Cε2) ₂ and that of Fcε3-4 (the region containing only the Cε3 and Cε4 domains), is found to become even sharper (62° to 54°) in the crystal structure IgE-Fc bound to sFcεRIα, the soluble extracellular domains of the IgE-binding receptor α chain ⁸ . Recent FRET studies with N‐ and C‐terminally tagged IgE‐Fc confirmed this increase in bending upon sFcεRIα binding ⁹ .

The FcεRI binding site spans both Cε3 domains in the region proximal to Cε28,10, although the Cε2 domain is not directly involved; the involvement of both chains determines the 1:1 binding stoichiometry. In contrast, two CD23 molecules bind to IgE-Fc, one for each chain and at the other, Cε4-proximal end of the Cε3 domain ^11,12,13 . CD23 binding also causes a conformational change in IgE-Fc ¹⁴ , but not one that greatly affects bending ⁹ . However, the relatively “closed” arrangement of the Cε3 domains in complex with the soluble CD23 head domain (sCD23), compared with free IgE-Fc, is inconsistent with the more “open” arrangement of these domains required for FcεRI binding. This partly explains the mutual exclusion of FcεRI and CD23 binding ^{11 , 12} , although it is likely that other factors, such as local conformational changes and modifications of conformational dynamics ¹⁵ , also contribute to the allosteric interaction between the two receptor binding sites ² .

A higher degree of flexibility in IgE-Fc was recently discovered by studying a complex with an anti-IgE-Fc Fab called aεFab ¹⁶ . Two aεFab molecules bind to IgE-Fc in a symmetrical manner, one to each Cε3 domain, adopting a fully extended conformation in which the local dual axes of the (Cε2) ₂ domains and the Fcε3-4 region are virtually identical. Analysis of complex formation in solution, along with molecular dynamics simulations of free IgE-Fc, suggested that the (Cε2) ₂ domain pair can “flip” from one side of the Fcε3-4 region to the other ¹⁶ . The IgE-Fc conformation stabilized by this anti-IgE antibody is incompatible with FcεRI binding, which explains its inhibitory activity.

Omalizumab is an anti‐IgE monoclonal IgG1 antibody that is approved for therapeutic use (Xolair ^® , Novartis) ¹⁷ . It binds to free IgE and inhibits the binding of both FcεRI and CD23; the binding site has been mapped to the Cε3 domain by peptide inhibition and molecular modeling methods, ^18,19 but the mechanism of action remains unknown. However, binding to FRET-labeled IgE-Fc indicates a small degree of extension ⁹ and therefore the possibility of allosteric rather than direct inhibition.

Recently, a type of inhibitor was discovered that actively disrupts preformed IgE/FcεRI complexes: Designed Ankyrin Repeat Protein (DARPin) was found to bind to the Cε3 domain of receptor-bound IgE and accelerate its dissociation from FcεRI ²⁰ . The crystal structure of the 2:1 complex of DARPin E2_79 with the Fcε3-4 molecule, bounded by an artificially created disulfide bond, made it possible to clarify the nature and location of the binding site, but the mechanism of action remained unknown. It was subsequently reported that omalizumab can similarly promote the dissociation of FcεRI-bound IgE, albeit at very high concentrations that are significantly higher than those that can be achieved with therapeutic use ^{21 , 22} .

Herein, we provide the crystal structure of a complex between IgE-Fc and a novel antibody fragment, Fab, derived from omalizumab (Omalizumab Fab3), which has three point mutations distal to the antigen-binding (IgE-Fc) complementarity determining regions (CDRs). . The mutations are S81R, Q83R and L158P with reference to SEQ ID NO:125 (or S77R, Q79R and L154P with reference to SEQ ID NO:129). The structure of the complex provides insight into the mechanism of action of omalizumab, and solution studies indicate that the mechanism is based on the intrinsic dynamics of IgE.

results

Despite great efforts, attempts to crystallize IgE-Fc in complex with omalizumab Fab resulted in selective crystallization of only the Fab fragment. Other authors have also reported similar unsuccessful attempts to crystallize this complex ²³ . Consequently, we constructed a new antibody, an omalizumab-derived Fab, with three point mutations, two in the framework region of the V _L domain (Ser81Arg, Gln83Arg) and one in the Cκ domain (Leu158Pro) (SEQ ID NO:125, PDB numbering) ( Figure 1) to disrupt the favorable crystal contacts observed in the omalizumab Fab crystal structure (results described elsewhere). We named this omalizumab-derived Fab fragment “Omalizumab Fab3.”

General structure of the IgE-Fc/Fab3 complex of omalizumab

We determined the crystal structure of the complex between IgE-Fc and Fab3 of omalizumab to 3.7 Å resolution (Figure 2A). Two Fab3 molecules of omalizumab (Fab ¹ and Fab ² ) bind to an asymmetric, partially curved IgE-Fc molecule, with each Fab binding one Cε3 domain (Figure 2B-C). Fab ¹ binds the Cε3 domain of IgE-Fc chain B, while Fab ² binds the Cε3 domain of IgE-Fc chain A. Due to the partially curved conformation of IgE-Fc in the complex, the Fab ² light chain also interacts slightly with the Cε2 domain of IgE-Fc chain B (more details on this interaction are provided later in this example).

Contact boundary between IgE-Fc and Fab3 of omalizumab

Each Fab3 omalizumab molecule binds one edge of the exposed surface of the Cε3 domain (chains C, C', F and G, and the base of the FcεRI receptor binding loop FG). Both the Fab3 heavy and light chains of omalizumab are involved, with the former responsible for ~60% of the ~715 Å ² contact area (Figures 2 and 3).

The Fab3 heavy chain contacts of omalizumab (SEQ ID NO:5), which differ slightly between the two contact surfaces, can be briefly described as follows: Gly32 and Tyr33 (CDRH1) enter into van der Waals interactions with Ala377 and Ser378 (Cε3) (sequence IgE-Fc is shown in SEQ ID NO:108 and Fig. 16), while Tyr54 (CDRH2) contacts Gly379-Pro381 (Cε3). The CDRH3 residues contribute the largest contact area and undergo a significant conformational change upon complex formation compared with unbound Fab structures (unpublished results ^19,23 ). The Ser100, His101, Tyr102, and Trp 106 residues of CDRH3 all engage in van der Waals interactions with residues of the Cε3 domain, which include Ser375-Gly379, Gln417, and Arg419 (Cε3). However, the most striking feature of this part of the contact surface is the interaction with Phe103 (CDRH3). Phe103 is mainly embedded in a pocket created by Thr373, the backbone of Trp374, Ser375, Gln417, and Arg419 (Cε3), and enters into a cation/π stacking interaction with Arg419 (Figure 3).

Arg419 (Cε3) also plays a key role in the interaction with the Fab3 light chain of omalizumab (SEQ ID NO:125) (Figure 3). Arg419 (Cε3) is within hydrogen bonding distance from the carbonyl oxygen atoms of the main chains of Tyr31 (CDRL1) and Asp32 (CDRL1), in addition to contacting the side chains of Asp32, Asp34 and Tyr36 (forming a hydrogen bond with the hydroxyl group of Tyr36). Asp32 also makes van der Waals interactions with Thr373 and Thr421 (Cε3). In contrast, only two residues of CDRL2 are involved in the contact interface: Tyr53 (CDRL2) contacts Gln417 (Cε3), and both Tyr53 and Tyr57 enter into van der Waals interactions with Met430 (Cε3); Tyr57 also forms a hydrogen bond with the Met430 backbone. In terms of heavy chain interactions, there are slight differences in light chain contacts between Fab ¹ and Fab ² .

Contact CDR residues on Fab3 of omalizumab; numbering in format (pdb/Kabat/Chothia). Heavy chain sequence: SEQ ID NO:5; light chain sequence: SEQ ID NO:125

CDRH1: Ser (31/31/31), Gly (32/32/31a), Tyr (33/33/32)

CDRH2: Tyr (54/53/53)

CDRH3: Ser (100/96/96), His (101/97/97), Tyr (102/98/98), Phe (103/99/99), Trp (106/101B/101B)

CDRL1: Asp (30/27C/30), Tyr (31/27D/30A), Asp (32/28/30B), Gly (33/29/30C), Asp (34/30/30D), Tyr (36 /32/32)

CDRL2: Tyr (53/49/49), Ser (56/52/52), Tyr (57/53/53), Ser (60/56/56)

CDRL1 and CDRH3 have the largest number of residues involved in the interaction and thus determine how omalizumab binds and targets IgE-Fc. CDRL3 is not involved in binding to IgE-Fc.

Comparison of the Fab3 interface of omalizumab with other anti-IgE complexes

The binding sites on the Cε3 domain for Fab3 of omalizumab and the recently described DARPin E2_79 ²⁰ overlap (Figure 4) and have similar sizes: ~715 Å ² and ~753 Å ² , respectively. Residues of the Cε3 domain shared by the two interfaces include Ser375-Gly379, Gln417, Arg419, Arg427, and Met430, but while omalizumab Fab3 makes closer contacts with the FG receptor binding loop of Cε3, the DARPin E2_79 interface continues in in the opposite direction, including the Cε3-4 domain linker.

The overlapping binding sites of Fab3 of omalizumab and DARPin E2_79 are markedly different from the interface recently described for Fab3 of omalizumab, which captures IgE-Fc in a fully extended conformation ¹⁶ (Figure 5). Not only is the contact surface area of omalizumab Fab3 approximately twice that of omalizumab Fab3 and DARPin E2_79, ~1400 Å ² , but omalizumab Fab3 binds IgE-Fc at a site centered on Arg393 in Cε3 and also contacts residues in the Cε2 domain and linker Cε2-Cε3 ¹⁶ . The crystal structure of another anti‐IgE Fab antibody, MEDI4212, in a 2:1 complex with Fcε3‐4 revealed another antibody binding site with the Cε3 domain, this site including an N‐linked oligosaccharide moiety at Asn394 ²⁴ .

IgE-Fc adopts a partially curved conformation upon binding to Fab3 of omalizumab

IgE-Fc in solution is predominantly in a bent state ^{5,6,9,25,26,27,28} and the crystal structure of free IgE-Fc exhibited a sharply bent (62°), asymmetric conformation in which a pair of domains (Cε2) ₂ fold back onto the Cε3 and Cε4 domains (Fig. 5A–B), with the Cε2 domain of one strand (chain B) contacting the Cε4 domain of the other (chain A) ^7,8 . IgE-Fc becomes even more sharply bent (54°) upon FcεRIα binding ^8,9 and associated conformational changes include angular displacement of the Cε3 domain of chain A, together with a pair of (Cε2) ₂ domains, in a rigid block, further away from the Cε3 domain chains B ⁸ .

In contrast to the complex with Fab3 of omalizumab, in which IgE-Fc adopts a fully extended, linear conformation, ¹⁶ IgE-Fc adopts a partially curved conformation when complexed with Fab3 of omalizumab (Figures 2C and 5C-D), consistent with previous FRET results. -assays that showed that omalizumab induces extension of IgE-Fc ⁹ . The site to which Fab ¹ binds is exposed in the free, sharply curved IgE-Fc, but further extension of the IgE-Fc, to just over 90°, is required for the site occupied by Fab ² to become accessible. This extension of IgE-Fc in complex with Fab3 of omalizumab is associated with the opening of both Cε3 domains, resulting in the creation of a nearly symmetrical Fcε3-4 region ( Fig. 2B ). The domain pair (Cε2) ₂ is located between the Cε3 and Cε4 domains from each chain, and is no longer associated as closely with the Cε3 domain from chain A.

Recent molecular dynamics simulations that examined the extension of IgE-Fc into an extended structure revealed that although the sharply bent conformation observed in the crystal structure of free IgE-Fc occupies the lowest energy level, another, distinct and defined energy level can be observed. level corresponding to the partially curved conformations of IgE-Fc ¹⁶ . The partially curved conformation adopted by IgE-Fc in the omalizumab/IgE-Fc Fab3 complex occupies this particular energy level (Figure 6).

Cε3 domains adopt a distinctly open conformation in the omalizumab/IgE-Fc Fab3 complex

In the crystal structures of the IgE-Fc and Fcε3-4 subfragment, the Cε3 domains adopt a range of different orientations ^{7,8,10,11,13,14,16,24,29} , a property associated with allosteric regulation of IgE binding to its two major receptors , FcεRI and CD23 ^8,11,12,14 . Both the distance between the Cε3 domains and their position relative to the Cε4 domains have been used to describe the variety of conformations observed for the Fcε3-4 region ²⁹ (a full description of these measurements is provided later in this example). In the omalizumab/IgE-Fc Fab3 complex, the Cε3 domains are located farther apart from each other and from the Cε4 domains than in any other crystal structure containing IgE-Fc or Fcε3-4, and thus adopt the most open conformation known in present (Fig. 5); this conformation is significantly more open than the conformation of FcεRI-bound IgE-Fc (Fig. 7).

Effect of Fab3 omalizumab on FcεRI and CD23 receptor binding

Omalizumab inhibits not only the interaction between IgE‐Fc and FcεRI, but also the interaction between IgE‐Fc and CD23 ³⁰ . Consistent with this, comparison of the omalizumab/IgE-Fc Fab3 and CD23/Fcε3-4 complexes ¹¹ revealed competition between omalizumab Fab3 and CD23 at both CD23 binding sites on Fcε3-4. Moreover, residues Arg376, Ser378 and Lys380 of the Cε3 domain are involved in the binding of both omalizumab Fab3 and CD23 ^{11 , 31} .

In contrast to CD23 binding to IgE, FcεRIα binds through both Cε3 domains. However, in the omalizumab/IgE-Fc Fab3 complex, the Cε3 domains adopt a conformation that is too open to allow simultaneous binding of both chains (Figure 8). Moreover, if the Fab3 omalizumab/IgE-Fc and sFcεRIα/IgE-Fc complexes are superimposed onto each of the Cε3 domains in turn, potential steric clashes occur in each case. When superimposed on the Cε3 domain, Fab ² Fab3 chains A of omalizumab would collide with the domain pair (Cε2) ₂ in complex with FcεRIα, and the Cε2-Cε3 linker from the complex with Fab3 of omalizumab would potentially collide with FcεRIα. When applied to the Cε3 domain of chain B, there would potentially be a collision of both Fab ¹ Fab3 of omalizumab and the Cε2-Cε3 linker (from the complex with Fab3 of omalizumab) with FcεRIα - although the binding sites for Fab ¹ and FcεRIα do not actually overlap.

However, the CDRL1 Fab3 residues of omalizumab are located in close proximity to the FcεRIα-binding loop of the FG Cε3 domain. This loop in strand B is involved in a hydrophobic proline sandwich interaction in which Pro426 in Cε3 is located between two tryptophan residues in FcεRIα. Asp32 (CDRL1) contacts Thr421, Gly33 (CDRL1) contacts Pro426, Arg427 and Ala428, and Asp34 (CDRL1) contacts Arg427 and Ala428. These interactions result in a change in the position of the FG loop of the Cε3 domain and would further interfere with the binding of IgE to FcεRI.

Binding of omalizumab to FcεRIα‐bound IgE has recently been reported ^{31 , 32} , although it is difficult to imagine how omalizumab could bind FcεRI‐bound IgE based on static crystal structures of IgE‐Fc complexed with sFcεRIα ⁸ and Fab3 of omalizumab. Therefore, we studied the binding of omalizumab Fab3 to IgE-Fc and characterized the interaction between omalizumab Fab3 and the IgE-Fc/FcεRI complex. The results provided insight into the mechanism of action of omalizumab.

Interaction of Fab3 omalizumab with IgE-Fc in solution

We characterized the IgE-Fc/Fab3 interaction of omalizumab in two different ways, either by directly immobilizing Fab3 of omalizumab on the surface and binding the IgE-Fc, or by binding Fab3 of omalizumab to His-tagged IgE-Fc captured on the sensor surface in a SPR assay. The C-terminal His-tagged IgE-Fc construct was captured using an anti-His tag antibody (GE Healthcare), and the binding characteristics of Fab3 of omalizumab, intact omalizumab, and Fab of omalizumab were compared. Not surprisingly, in competitive binding experiments, all three molecules competed for the same binding sites and exhibited approximately similar binding affinities (data not shown). The omalizumab Fab3 construct had slightly higher affinity compared to omalizumab Fab3 and intact omalizumab (Figure 8A-C). According to the crystal structure, two Fab3 molecules of omalizumab bind to IgE-Fc: binding is clearly biphasic, with a high affinity interaction (~1 nM) observed at low ligand concentrations and a second (weaker) binding site (~30 nM), observed at higher concentrations (Figure 9A).

The SPR experiment in a sandwich format allowed us to separately characterize the two IgE-Fc/Fab3 binding sites of omalizumab. Using this approach, Fab3 of omalizumab was covalently immobilized on the sensor surface and IgE-Fc was passed over this surface. At low concentrations under these conditions, the high-affinity site dominates the interaction and the binding curves can be described in accordance with the kinetics of single-phase interaction (K _D ~ 1 nM, k _on ~ 1.2×10 ⁶ M ^-1 s ^-1 , k _off ~ 8 ×10 ^-4 s ^-1 ). This 1:1 IgE-Fc/Fab3 complex of omalizumab, captured on the biosensor surface by SPR, can then be used to measure the binding of a second omalizumab Fab3 molecule, the binding of which is significantly weaker (K _D ~ 30 nM, k _on ~ 2 × 10 ⁵ M ^-1 s ^-1 , k _off ~ 6×10 ^-3 s ^-1 ) than the first (Fig. 9B).

Competition between Fab3 of omalizumab and FcεRIα for binding sites and formation of the Fab3 omalizumab/IgE-Fc/FcεRIα complex

We then examined the ability of omalizumab Fab3 to influence the interaction between IgE-Fc and FcεRIα. In solution competition binding experiments, Fab3 omalizumab at increasing concentrations inhibited the binding of IgE-Fc to FcεRIα (Figure 8D). Mechanistically, Fab3 of omalizumab influences both the number of available binding sites (B _max ) and the apparent K _D of IgE-Fc/FcεRIα interaction; this is characteristic of a mixed mechanism of inhibition ³³ . A decrease in B _max values is an indicator of the process of allosteric inhibition, and a decrease in apparent interaction affinity is most often associated with direct competition for a common binding site (i.e., orthosteric inhibition), but can also be observed for some allosteric inhibitors. Considering the binding sites observed in the crystal structure, it is possible that Fab3 of omalizumab inhibits IgE-Fc binding to FcεRI through both orthosteric and allosteric mechanisms.

Competition between omalizumab and FcεRIα for binding sites has been described in many publications, but has always been interpreted as direct competition for the same (or overlapping) binding site. This interpretation has often been used to explain why omalizumab fails to bind to IgE-FcεRI complexes on cells. However, we found that Fab3 of omalizumab can bind with high affinity to IgE-Fc that is already bound to FcεRIα (Figure 9C, inset). Other studies have suggested the existence of an omalizumab/IgE-Fc/FcεRI complex ^31,32 but this complex has not previously been experimentally characterized. The data suggest that although IgE-Fc binding to FcεRIα did not result in a large change in the affinity of omalizumab Fab3 for IgE-Fc, it did result in a marked change in the number of available binding sites for omalizumab Fab3 in the population of FcεRIα-bound IgE-Fc molecules. We compared the binding K _D and _Bmax values for an IgE-Fc molecule captured via an anti-His marker antibody with those for a molecule captured by sFcεRIα and found that FcεRIα-bound IgE-Fc had less than 10% of the omalizumab Fab3 binding sites compared to IgE-Fc captured via the His marker, which, as expected, exhibited binding levels consistent with a 2:1 stoichiometry (Figure 9C). Thus, contrary to what was previously suspected, omalizumab does not bind to mast cell-bound IgE because the FcεRIα and omalizumab binding sites overlap. Instead, FcεRIα affects IgE-Fc allosterically by altering the dynamic equilibrium of different IgE-Fc conformations ¹⁶ , resulting in a significant reduction in the number of omalizumab binding sites in the population of FcεRIα-bound IgE-Fc molecules.

Mechanism of omalizumab Fab3-mediated accelerated dissociation of the IgE-Fc/FcεRIα complex

The publication by Kim et al. ²⁰ reported that DARPin E2_79 can accelerate the breakdown of previously formed IgE–FcεRI complexes. In continuation of these results, Eggel et al. ²¹ it was later demonstrated that omalizumab can also stimulate the dissociation of IgE from FcεRI. Likewise, we found that when omalizumab Fab3 is associated with the IgE-Fc/FcεRIα complex, it can accelerate the dissociation of IgE-Fc from FcεRIα (Figure 9D), and that omalizumab Fab3 does this more efficiently than omalizumab Fab3 and much more efficiently. more effective than intact omalizumab (Figure 10E). One Fab binds to the IgE-Fc/FcεRIα complex but does not accelerate the dissociation of IgE-Fc from FcεRIα. Strikingly, it appears that accelerated dissociation occurs only after binding to the second binding site (i.e., the low-affinity site). The tetramolecular complex (Fab3 of omalizumab) ₂ /IgE-Fc/FcεRIα should change the energy environment of IgE-Fc so that the energy barrier to dissociation of IgE-Fc/FcεRIα is markedly reduced, resulting in rapid dissociation of an otherwise very stable complex.

Details of the interaction between the Fab3 light chain of omalizumab and the Cε2 domain

In the omalizumab/IgE-Fc Fab3 complex, one Cε2 domain interacts only slightly over an area of approximately 260 Å ² (compared to an average interaction area of ~715 Å ² between omalizumab Fab3 and the Cε3 domain) with two of the mutant residues (Ser81Arg and Gln83Arg ). There is no contact between Pro158 and IgE-Fc.

The Arg81 side chain from the Fab ² light chain (one of the mutated residues in Fab3 of omalizumab; (SEQ ID NO:125, PDB numbering)) folds opposite the Val277 and Asp278 Cε2 domain from chain B (SEQ ID NO:108). Ser80 (Fab3 of omalizumab) folds opposite Asp278, Leu279 and Thr281 (Cε2 domain), while Ser64 (Fab3 of omalizumab) folds opposite Asp276 and Asp278. Ser64 and Ser80 are identical in omalizumab and Fab3 of omalizumab.

In the omalizumab/IgE-Fc Fab3 complex, Arg83 (one of the mutated residues in the omalizumab Fab3) does not appear to contact the Cε2 domain due to a disruption in the side chain of Asp278 (Cε2 domain). However, if the side chain of Asp278 were intact, a hydrogen bond or salt bridge could potentially form between Arg83 and Asp278.

Contacts in the Fab3 omalizumab/IgE-Fc complex determined by crystallography

Contacts between antibody and antigen within 4 Å of a crystal structure are typically characterized by an epitope/paratope interface.

IgE-Fc residues within 4 Å of the Fab3 heavy and light chain CDR regions of omalizumab define the following epitope:

T 373, W 374, S 375, R 376, A 377, 3 S 78, G 379, P 381, Q 417, C 418, R 419, P 426, R 427, A 428 (on chain A).

In addition, IgE-Fc residues within 4 Å of residues FR1 and FR3 of the Fab3 light chain of omalizumab extend the epitope to:

D 278, T 281 (on chain B - Cε2 domain) [with contacts with R18, S64, S80 and R81 of the antibody - and in addition R83, as found in the molecular dynamics simulation described in Example 5].

Contacts between antibody and antigen within 5 Å in the crystal structure are also informative for determining the antibody/antigen contact surface.

Additional IgE-Fc residues within 5 Å of the Fab3 heavy and light chain CDR regions of omalizumab are: K380, M430 (on chain A).

In addition, additional IgE-Fc residues within 5 Å of residues FR1 and FR3 of the omalizumab Fab3 light chain are: D276, V277, L279, S280, A282 (on chain B - Cε2 domain) [with additional contacts with G16 in FR1 and R65 to FR3].

Analysis of the orientation of the Cε3 domain in the omalizumab/IgE-Fc Fab3 complex

In one method for analyzing the position of Cε3 domains relative to Cε4 domains, the interatomic distance between the Cα atom of Asn394 of a Cε3 domain from one chain and the Cα atom of Lys497 of a Cε4 domain of another chain was used to describe the “openness” of Cε3 domains ²⁹ . The interatomic distance between Cα Val336 has been used to describe the “span,” or how close the Cε3 domains are to each other ²⁹ .

For FcεRI-bound IgE-Fc and FcεRI-bound Fcε3-4, in which the Cε3 domains adopt an open conformation, the "open" values are in the range of 23.5-28.4 Å, while the "span" values are in average 23.3 Å ^8.10 . The corresponding values for the omalizumab/IgE-Fc Fab3 complex average 29.5 for “open” and 29.4 Å for “sweep.” When complexed with Fab3 of omalizumab, the Cε3 domains adopt the most open conformation (furthest apart) described to date.

Discussion

We report the 3.7 Å resolution structure of a complex between IgE-Fc and a Fab fragment derived from the therapeutic anti-IgE antibody omalizumab; We refer to this Fab fragment containing three point mutations in framework regions distal to the antigen binding site as omalizumab Fab3. The structure shows two Fab3 molecules of omalizumab complexed with IgE-Fc, one bound to each of the two Cε3 domains (but only one of the Fabs bound to the Cε2 domain), and provides insight into the ability of omalizumab to inhibit IgE binding to both FcεRI. so with CD23. IgE-Fc was also found to adopt a partially curved conformation when complexed with Fab3 of omalizumab, consistent with our earlier studies using FRET-labeled IgE-Fc, which indicated a slight extension of relatively free IgE-Fc ⁹ .

In solution, IgE-Fc is predominantly in a bent state ^{5,6,9,25,26,27,28} , and in the crystal structure of free IgE-Fc, a pair of domains (Cε2) ₂ are folded back opposite the Cε3 and Cε4 domains ^7,8 . Recently, a greater understanding of the conformational flexibility of IgE-Fc was achieved when we solved the structure of the fully extended conformation that occurs in complex with anti-IgE-Fc Fab (aεFab) ¹⁶ . Molecular dynamics simulations examining the extension of IgE-Fc from a fully bent to an extended conformation revealed energy levels corresponding to partially bent conformations (Figure 6). The omalizumab/IgE-Fc Fab3 complex described herein, in which the bend between the (Cε2) ₂ domain pair and the Fcε3-4 domains is ~90°, corresponds to a specific energy level in this simulation ¹⁶ . Interestingly, the location of the Fab3 binding site of omalizumab would not prevent further extension to a fully extended conformation, and therefore it is possible that IgE-Fc could undergo significant changes in conformation even when complexed with omalizumab.

In addition to the bending of the (Cε2) ₂ domain pair relative to the Cε3 and Cε4 domains, different structures of the IgE-Fc, Fcε3-4 and receptor complex have shown that the Cε3 domains can adopt a range of relative orientations, from closed to open ^{7,8,10. 11,13,14,16,24,29} . The opening and closing of Cε3 domains plays a role in the allosteric regulation of IgE-Fc receptor binding ^11,12 : in complex with CD23 they are relatively closed ^11,13,14 , while in complex with FcεRI they are more open ^8,10 . Comparison of the structures of the CD23/Fcε3-4 and Fab3 omalizumab/IgE-Fc complexes shows that the CD23 and omalizumab binding sites overlap, and competition binding experiments indicate that the inhibition of IgE binding to CD23 by omalizumab is clearly orthosteric.

However, inhibition of FcεRI binding is more complex. When complexed with Fab3 of omalizumab, the Cε3 domains adopt a more open conformation than could be seen in the previously known structure, so much so that the two interaction subsites between IgE-Fc and FcεRI, one involving each Cε3 domain, cannot bind simultaneously. Another contribution to this inhibition may result from the proximal location of the omalizumab Fab3 molecule (Fab ¹ ) to the FG receptor-binding loop of Cε3, which may directly influence the conformation of contact residues for FcεRIα. Finally, even if the Fab3 binding epitopes of omalizumab and FcεRIα on IgE-Fc are not strictly overlapping, there is the potential for steric clashes if both bind simultaneously. Thus, the crystal structure suggests that the mechanism of inhibition of omalizumab is primarily allosteric, but with a possible orthosteric component.

SPR studies allowed the inventors to evaluate the kinetics and affinity parameters for the two Fab3 binding sites of omalizumab. The two affinity values differed markedly, with K _D values of ~1 nM and ~30 nM, the former of which is associated with a higher association rate constant (k _on ~ 2×10 ⁶ M ^-1 s ^-1 versus ~ 2×10 ⁵ M ^-1 s ^-1 ) and a slightly lower dissociation rate constant (k _off ~ 2×10 ^-3 s ^-1 compared to ~ 6×10 ^-3 s ^-1 ). It can be reasoned that the higher affinity interaction corresponds to the binding of Fab ¹ , which would have unimpeded access to the curved IgE-Fc molecule, while the lower affinity and lower rate of association are characteristic of Fab ² , but the inventors cannot this can be stated with confidence.

Additional SPR experiments to study the mechanism of inhibition of IgE-Fc binding to FcεRIα by Fab3 of omalizumab demonstrated a decrease in the number of accessible sites for Fab3 of omalizumab on IgE-Fc (reduction _{in Bmax} ) complexed with FcεRIα. Inhibition of IgE binding to FcεRI by omalizumab has often been interpreted as direct competition for overlapping sites, however there have been reports that omalizumab can bind to receptor‐bound IgE ^{21 , 32} . We directly demonstrated the ability of omalizumab Fab3 to bind IgE-Fc that is already bound to FcεRIα to form a trimolecular complex. The effect of prebinding IgE-Fc to FcεRIα is to reduce the number of omalizumab Fab3 binding sites on IgE-Fc to less than 10% of the number of sites available in free IgE-Fc; such an effect can only be a consequence of allosteric modulation.

The nature of the interaction of omalizumab Fab3 with the IgE-Fc/FcεRI complex provides insight into the mechanism of accelerated dissociation. This phenomenon was first reported for DARPin and then for omalizumab, ^20,21 in the latter case at significantly higher concentrations than those used in therapy, ²² and is now described herein for Fab fragments of omalizumab. We also demonstrated that dissociation occurs only after the first binding of the second (lower affinity) Fab3 molecule of omalizumab. In other words, a tetramolecular complex - (Fab3 omalizumab) ₂ /IgE-Fc/FcεRIα - must be formed for a significant acceleration of dissociation to occur.

Based on the inventors' observations for Fab3 of omalizumab, IgE-Fc and sFcεRIα, they propose the following mechanism for the interaction of omalizumab, IgE and FcεRI: IgE binds to FcεRI and, under these conditions, a small population of such bound IgE molecules adopt a conformation to which the molecules can bind omalizumab; When the second omalizumab molecule binds to form a tetrameric complex, the energy environment of IgE changes such that the interaction with FcεRI is destabilized and rapid dissociation of IgE from FcεRI occurs. The key to understanding this mechanism is to recognize the complexity of the energy environment for IgE and that different conformational states exist in dynamic equilibrium.

The inhibitory activity of omalizumab appears to be related to the characteristic flexibility of IgE and, at least in the case of the accelerated dissociation process, to the dynamics of the IgE/FcεRI complex. IgE has a number of unusual structural characteristics compared to antibodies of other isotypes, including the presence of Cε2 domains and the unique conformational dynamic, molten globule-like character of the Cε3 domains ³⁴ . Collectively, these properties create an allosteric interaction pathway that prevents simultaneous binding of CD23 and FcεRI; this is important to avoid allergen‐independent mast cell activation resulting from cross‐linking of FcεRI‐bound IgE by the trimeric CD23 molecule ¹² . Other functional advantages associated with IgE dynamics have been proposed for the membrane‐bound B‐cell IgE receptor ¹⁶ . The observation that omalizumab does not use the expected orthosteric mechanism to inhibit the IgE/FcεRI interaction indicates that it exploits the unusual dynamic properties of IgE, both as a blocking antibody and in terms of its ability to avoid cross-linking with IgE-bound mast cells. Finally, omalizumab can actively induce the dissociation of IgE from FcεRI, albeit at concentrations higher than therapeutic ²¹ , due to allostery and the inherent flexibility of IgE, which is retained even in complex with its receptors.

Methods

Protein cloning, expression and purification. Omalizumab human IgG ₁ and omalizumab Fab3 were cloned, expressed, and purified as described in ¹⁶ . IgE-Fc was prepared as previously described ³⁵ . IgE-Fc has SEQ ID NO:108 (V224-K547 according to Dorrington & Bennich (1978) Immunol. Rev. 41:3-25, but with the following mutations introduced into IgE-Fc to simplify the glycosylation pattern: N265Q and N371Q ). Omalizumab was purchased from Novartis Europharm Limited. The 2:1 Fab3 complex of omalizumab/IgE-Fc was purified by size exclusion chromatography, eluted in 25 mM Tris-HCl, pH 7.5, 20 mM NaCl and 0.05% (w/v) NaN ₃ , and concentrated to 23 mg/ ml.

Surface plasmon resonance. SPR experiments were performed on a Biacore T200 instrument (GE Healthcare). Specific surfaces were prepared either by covalently cross-linking proteins using an amine binding protocol (GE Healthcare), with a binding density of <300 response units, or by capturing His-tagged proteins using an anti-His sensor surface. To capture His-tagged ligands, a monoclonal anti-His marker antibody was used and immobilized according to the manufacturer's instructions (Biacore His Capture Kit, GE Healthcare). In binding experiments, association times of 180–240 s were typically used, and component dissociation was monitored for at least 500 s. Injections were performed at a flow rate of 25 μl min ^-1 in running buffer 20 mM HEPES, pH 7.4, 150 mM NaCl and 0.005% (v/v) P-20 surfactant (GE Healthcare). Most experimental measurements were performed at 25°C; Some sandwich binding experiments were performed at 5°C to minimize accelerated dissociation phenomena. Standard double reference subtraction methods were used ³⁶ and kinetic curves were generated using the Origin program (OriginLab).

TR-FRET. IgE-Fc was labeled with a fluorophore donor by reacting the protein at a concentration of 4 mg/ml in a solution of 100 mM sodium bicarbonate, 50 mM NaCl, pH 9.3, with a 5-fold molar excess of terbium isothiocyanate chelate (Invitrogen). After 3 hours of incubation at room temperature with stirring, excess unreacted fluorophore was removed by dialysis in PBS (20 mM phosphate-buffered saline, 150 mM NaCl, pH 7.4). The sFcεRIα-IgG ₄ -Fc(α-γ) ³⁷ fusion protein was labeled with a fluorophore acceptor by reacting the protein at a concentration of 3 mg/ml with a 2.5-fold molar excess of Alexa Fluor 647 succinimidyl ester (Invitrogen) for 1 hour at room temperature . Excess fluorophore was removed by dialysis in PBS.

TR-FRET competitive inhibition assays were performed using 1 nM terbium-labeled IgE-Fc and 0-20 nM Alexa Fluor 647-labeled sFcεRIα-IgG ₄ -Fc with Fab3 of omalizumab over a range of concentrations. Assays were performed in HiBase 384-well white plates (Greiner BioOne) using Lanthascreen buffer (Invitrogen) as diluent. The plate was left to incubate overnight at room temperature and readings were taken on an Artemis plate reader (Berthold Technologies). TR-FRET ratios were then calculated for each well as the acceptor emission at 665 nm divided by the donor emission at 620 nm multiplied by 10,000.

Crystallization. Crystals with a rectangular morphology, up to 400 μm in length, were grown at 18°C using the sessile drop vapor diffusion method. The reservoir contained 50 μL of 4% (w/v) PEG 8000 and 0.03 M sodium fluoride, and the drop contained 100 nL of protein and 300 nL of reservoir. Despite significant optimization efforts, the diffraction quality of the crystals could not be improved beyond what was achieved in this study. The crystals typically began to grow within a few days, and often dissolved in their droplets, but could be stabilized in 4 M TMAO (trimethylamine N-oxide), which has been successfully used as a cryoprotectant.

Data collection and processing. Data were collected using locked beams I02 and I03 on a Diamond light source (Harwell, UK). Integration was performed using the XDS ³⁸ option in the xia2 ³⁹ expert system. The crystals exhibited anisotropic diffraction, and data from several crystals were combined. Data were scaled to 3.7 Å resolution using AIMLESS from the CCP4 suite ^40,41 and then trimmed to resolution limits of 3.7 Å (a*), 3.9 Å (b*), and 4.2 Å (c*) using the UCLA Anisotropic Diffraction Server ⁴² . Calculation of the Matthews coefficient showed a solvent content of ~62% for a single 2:1 Fab3 omalizumab/IgE-Fc complex (MW ~170 kDa) in the asymmetric unit.

Definition of structure, construction and refinement of the model. The structure was solved by molecular replacement using the programs PHASER ⁴³ and MOLREP ⁴⁴ from the CCP4 package ⁴⁰ , using protein atoms from PDB entry 2wqr ⁸ and the Fab structure of omalizumab at 1.9 Å resolution (unpublished results) as search models. Refinement was initially performed using REFMAC ⁴⁵ and then PHENIX ⁴⁶ , and alternated with manual model building in Coot ⁴⁷ . Model quality was assessed using MolProbity ⁴⁸ , POLYGON ⁴⁹ and other validation tools in the PHENIX GUI ⁵⁰ . Statistics for data processing and refinement are presented in Table 1. The electron density map area is shown in Fig. 11. Contact surfaces were analyzed using PISA ⁵¹ and figures were generated using PyMOL ⁵² .

Table 1. Statistics of data processing and clarification

Data processing Space group I 2 ₁ 2 ₁ 2 ₁ Unit cell dimensions (Å) a=76.64, b=231.19, c=247.12 Resolution (Å): general (outer shell) 115.59-3.70 (4.10-3.70) Completeness (%) ^a 99.9 (99.9) Plurality ^a 38.0 (38.4) Average ((I)/σ(I)) ^a 17.9 (1.9) R _pim (%) ^a 2.6 (56.3) Clarification ^b R _work /R _free (%) ^c 25.88/30.92 Number of reflections 20,087 RMS Bond lengths (Å) 0.002 Tie angles (°) 0.451 Coordinate error (Å) 0.60 Avg. B-factor (Å ² ) 171.2 Ramachandran Map Preferred (%) 95.81 Acceptable (%) 100.00

^a Values in parentheses refer to the highest resolution layer

^b Refinement was performed with data trimmed to resolution limits of 3.7 Å (a*), 3.9 Å (b*) and 4.2 Å (c*)

^c R _free set includes 5% reflections.

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Example 2: Measurement of accelerated dissociation of IgE-Fc from immobilized sFcεRIα using Biacore

Biacore technology allows the measurement of interactions between biological molecules without the need for labeling. One of the interacting components, called the ligand, is either directly immobilized or trapped on the sensor surface, while the other, called the analyte, flows in solution above the retention surface. The sensor detects the change in mass on the sensor surface when the analyte binds to the ligand and when the analyte dissociates from the ligand. This corresponds to the processes of association and dissociation. In the accelerated dissociation assay, sFcεRIα is the ligand and is immobilized on the sensor surface. IgE-Fc is an analyte and is captured by sFcεRIα. Dissociation of IgE-Fc from sFcεRIα is controlled either by flow of buffer over the sensor surface or by flow of IgE binding partner solution over the sensor surface. Method details:

Device: Biacore 3000, GE Healthcare AB, Uppsala, Sweden

Touch chip: CM5. Catalog number BR100399

BIAnormalising solution: 70% (by weight) glycerin solution. Part of the BIAmaintenance suite. Catalog number BR100651. The BIAmaintenance kit was stored at 4°C.

Amino Bonding Kit: Catalog number BR100633. Ethyl 3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) was adjusted to 75 mg/mL in distilled water and stored in 200-μL aliquots at −70°C. N-Hydroxysuccinimide (NHS) was adjusted to a concentration of 11.5 mg/mL in distilled water and stored in 200-μL aliquots at −70°C. Ethanolamine hydrochloride-NaOH, pH 8.5, was stored at 4°C.

Reagent for sFcεRIα oxidation. Carbohydrazide (SigmaAldrich, catalog number C11006) was adjusted to a concentration of 5 mM in distilled water. Sodium cyanoborohydride (SigmaAldrich, cat. no. 156159) was adjusted to a concentration of 100 mM in sodium acetate (BDH, cat. no. S1104-500GM), 100 mM, pH=4. Sodium m-periodate (SigmaAldrich, cat. no. S-1878) was adjusted to 50 mM in sodium acetate (BDH, cat. no. S1104-500GM), 100 mM, pH=5.5.

sFcεRIα was diluted to a concentration of 1 mg/ml in 0.1 M sodium acetate, pH 5.5. Then, 4 μL of sodium periodate (50 mM, diluted 1/50) was added to 200 μL of 1 mg/mL sFcεRIα solution. The mixture was left on ice for 20 minutes. Before immobilization, the sFcεRIα solution was diluted to a concentration of 7 μg/ml with 10 mM sodium acetate solution (GE Healthcare, catalog number BR100669), pH=4.0.

Buffers: The running buffer is HBS-EP (namely, 10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% P20 surfactant, reconstituted from 10x stock solution): catalog number BR100669. The immobilization buffer is acetate 4.0 (namely, 10 mM sodium acetate solution, pH 4.0). Catalog number BR100349. The buffer was stored at 4°C.

Ligand: sFcεRIα, which is an extracellular fragment of the alpha chain of the human high-affinity IgE receptor. Expressed as a recombinant protein in CHO cells and purified.

Analyte: IgE-Fc, which is the Fc fragment of human IgE expressed as a recombinant protein in CHO cells and purified. Wild-type human IgE-Fc (domains Cε2-Cε4 numbered V224-K547 according to Dorrington & Bennich (1978) Immunol. Rev. 41:3-25) carrying the C225A mutation (SEQ ID NO:108) was used.

Mutant nomenclature:

- Omalizumab Fab1: S81R, Q83R [S77R, Q79R according to Kabat numbering] (light chain variable region+kappa constant region, SEQ ID NO:24, where S77 and S79 are replaced by Q; heavy chain variable region+CH1 constant region, SEQ ID NO:5)

- Omalizumab Fab2: L158P [L154P according to Kabat numbering] (light chain variable region+kappa constant region, SEQ ID NO:113; heavy chain variable region+CH1 constant region, SEQ ID NO:5)

- Omalizumab Fab3: S81R, Q83R, L158P [S77R, Q79R, L154P according to Kabat numbering] (light chain variable region, SEQ ID NO:121; heavy chain variable region+CH1 constant region, SEQ ID NO:5).

IgE binding partners (1): full-length omalizumab (Novartis); recombinant Fab fragment of omalizumab expressed in CHO cells and purified.

IgE binding partners (2): recombinant Fab fragment of omalizumab, with its mutations, expressed in HEK-293 cells and analyzed as culture supernatant. The culture supernatant was concentrated tenfold before analysis.

Assay method (1): sFcεRIα was bound to the sensor surface via aldehyde group binding to a level of ~500 response units (RU). HBS-EP buffer was used as running buffer at a flow rate of 30 μL/min. IgE-Fc was diluted to a concentration of 10 nM in HBS-EP and injected over immobilized sFcεRIα for 290 seconds, followed by 3 injections, each lasting 690 seconds, of running buffer or IgE binding partner diluted in running buffer. The level of IgE-Fc uptake was ~90 EU. The surface was regenerated with two 60-second injections of 10 mM glycine-HCl solution, pH 2.5. The amount of IgE-Fc dissociated from immobilized sFcεRIα was calculated as a function of the amount originally bound, and the rate of dissociation was calculated as the amount of IgE-Fc dissociated from immobilized sFcεRIα normalized to the amount initially bound, as a function of time elapsed.

Assay method (2): sFcεRIα was bound to the sensor surface via aldehyde group binding to a level of ~2000 response units (RU). HBS-EP buffer was used as running buffer at a flow rate of 30 μL/min. IgE-Fc was diluted to a concentration of 10 nM in HBS-EP and injected over immobilized sFcεRIα for 180 seconds, followed by 1 injection over 180 seconds of running buffer or IgE binding partner diluted in running buffer. The IgE-Fc uptake level was ~275 EU. The surface was regenerated with two 60-second injections of 10 mM glycine-HCl solution, pH 2.5. The amount of IgE-Fc dissociated from immobilized sFcεRIα was calculated as a function of the initially bound amount, and the dissociation rate was calculated as the amount of IgE-Fc dissociated from immobilized sFcεRIα normalized to the initially bound amount as a function of time elapsed.

IgE binding partner IgE-Fc uptake level (%) Amount of IgE-Fc remaining bound to sFcERIa after 1st injection (%) Amount of IgE-Fc remaining bound to sFcERIa after 2nd injection (%) Amount of IgE-Fc remaining bound to sFcERIa after 3rd injection (%) Apparent dissociation rate based on 3rd injection of IgE binding partner (1/s) Omalizumab 300 mcg/ml
buffer
Fab1 omalizumab 100 µg/ml
Fab2 omalizumab 100 µg/ml
Fab3 omalizumab 100 µg/ml 100.0
100.0
100.0
100.0
100.0 79.3
87.6
61.3
79.4
45.3 71.5
81.9
51.0
71.5
34.6 69.9
77.9
47.3
69.5
30.8 1.46E-04
1.02E-04
3.06E-04
1.48E-04
4.80E-04

Table 2: Calculation of the amount of IgE-Fc dissociated from immobilized sFcεRIα. The initial binding of IgE to sFcεRIα was set as 100%, and dissociation was calculated relative to this. The apparent dissociation rate is based on an assumed single-rate model.

Further mutagenesis was performed to determine whether additional mutations could be identified that lead to accelerated dissociation of IgE-Fc from sFcεRIα, in addition to those described for Fab3. As a result, the S64M mutation was identified (with reference to SEQ ID NO:20), which in the context of Fab3 was capable of leading to a further increase in the dissociation of IgE from sFcεRIα. These data are shown in Figure 13 and Table 3.

Sample Description Captured IgE-Fc, EO IgE-Fc after dissociation, EO % Dissociation Apparent dissociation rate (1/s) control, cells only control supernatant 256.19 215.2 16.00 7.03E-04 mutation S64M Omalizumab Fab (S64M, S81R, Q83R, L158P) 278.59 94.66 66.02 4.30E-03 Fab omalizumab DT Fab omalizumab DT 265.49 205.05 22.77 22.77 Fab3 omalizumab Omalizumab Fab (S81R, Q83R, L158P) 261.85 163.32 37.63 1.90E-03

Table 3: Calculation of the amount of IgE-Fc dissociated from immobilized sFcεRIα. The initial binding of IgE to sFcεRIα was set as 100%, and dissociation was calculated relative to this. The apparent dissociation rate is based on an assumed single-rate model. Mutant S64M: light chain SEQ ID NO:39; Omalizumab DT Fab: light chain SEQ ID NO:20; Fab3 of omalizumab: light chain SEQ ID NO:125. In all three, the heavy chain has SEQ ID NO:5.

Conclusion:

Taken together, our data indicate that a mutant Fab form of omalizumab can accelerate the dissociation of IgE from an immobilized form of the high-affinity IgE receptor, FcεRI. Light chain mutations that make this possible include, but are not necessarily limited to, S64M, S81R, Q83R and L158P with reference to SEQ ID NO:24, and the result is SEQ ID NO:39.

Example 3: Measurement of accelerated dissociation of IgE-Fc from FcεRI by flow cytometry

Device: FACSCanto II flow cytometer (Becton Dickinson)

Cell line: RBL-SX38 cells expressing human FcεRI were cultured in minimal maintenance medium supplemented with 10% fetal bovine serum, 2 mM GlutaMAX and 500 μg/ml geneticin (Life Technologies). During analysis, cells were washed in PBS and incubated in Accutase until detached, then resuspended at a density of 1×10 ⁶ cells/ml in culture medium. All subsequent stages of incubation were carried out in a culture medium.

Method of analysis: RBL-SX38 cells at a density of 1×10 ⁶ cells/ml were incubated with a 5 nM solution of Alexa 488-labeled IgE-Fc for 1 hour at 37°C. Cells were washed twice in culture medium to remove unbound IgE-488, then suspended at a density of 1×10 ⁶ cells/ml in culture medium, or IgE-binding fragments were diluted in culture medium at a concentration of 100 μg/ml. The cells were then incubated at 37°C with constant stirring. At each time point, 0.5 x 10 ⁵ cells were collected, washed in ice-cold PBS, and then fixed by resuspension in 1% paraformaldehyde in PBS for 16 hours at 4°C. The amount of cell-bound Alexa 488 fluorescence was determined using a FACSCanto II flow cytometer.

Flow Cytometry: Fixed cells were washed twice in FACS buffer (0.1% w/v BSA, 0.01% w/v NaN ₃ in PBS, pH 7.4) and resuspended in 200 μl FACS buffer. Flow cytometry was performed on a FACSCanto II cytometer using standard methods, and the geometric mean of the fluorescence intensity of Alexa 488 associated with intact cells was calculated using the FlowJo program. The rate of dissociation of Alexa 488-labeled IgE-Fc was calculated as the change in the geometric mean value of fluorescence intensity over time, as a result of incubation of cells either in a culture medium, an excess of unlabeled IgE-Fc, or in the presence of IgE-binding fragments.

Mutant nomenclature:

- Omalizumab Fab1: S81R, Q83R [S77R, Q79R according to Kabat numbering] (light chain variable region+kappa constant region, SEQ ID NO:24, in which S77 and S79 are replaced by Q; heavy chain variable region+CH1 constant region ,SEQ ID NO:5)

- Omalizumab Fab2: L158P [L154P according to Kabat numbering] (light chain variable region+kappa constant region, SEQ ID NO:113; heavy chain variable region+CH1 constant region, SEQ ID NO:5)

- Omalizumab Fab3: S81R, Q83R, L158P [S77R, Q79R, L154P according to Kabat numbering] (light chain variable region+kappa constant region, SEQ ID NO:121; heavy chain variable region+CH1 constant region, SEQ ID NO: 5).

IgE binding partner Concentration (µg/ml) Dissociation rate (x10 ^-5 ) 1/s BUT 1.38 Omalizumab 100 1.77 Omalizumab 31 1.56 Omalizumab 10 1.32 Omalizumab 3.1 1.45 Omalizumab 1 1.36 Omalizumab Fab 100 2.09 Omalizumab Fab 31 1.63 Omalizumab Fab 10 1.35 Omalizumab Fab 3.1 1.38 Omalizumab Fab 1 1.59 Fab1 of omalizumab 100 4.14 Fab1 of omalizumab 31 2.6 Fab1 of omalizumab 10 1.93 Fab1 of omalizumab 3.1 1.61 Fab1 of omalizumab 1 1.46 Fab2 of omalizumab 100 2.11 Fab2 of omalizumab 31 1.65 Fab2 of omalizumab 10 1.39 Fab2 of omalizumab 3.1 1.36 Fab2 of omalizumab 1 1.29 Fab3 omalizumab 100 3.99 Fab3 omalizumab 31 2.81 Fab3 omalizumab 10 2.03 Fab3 omalizumab 3.1 1.5 Fab3 omalizumab 1 1.47 Isotype control 100 1.3 Isotype control 31 1.28 Isotype control 10 1.35 Isotype control 3.1 1.19 Isotype control 1 1.25 IgE quenching 1.24

Table 4: Calculated rate of dissociation of Alexa 488-labeled IgE-Fc from the surface of RBL-SX38 cells. The apparent dissociation rate was determined by linear regression based on a plot of -ln(R1/R0) versus time for all available time points.

Conclusion:

Taken together, our data indicate that a mutant Fab form of omalizumab can accelerate the dissociation of IgE from the high-affinity IgE receptor, FcεRI, expressed on the cell surface. Light chain mutations that make this possible include, but are not necessarily limited to, S81R, Q83R, and L158P.

Example 4: Demonstration that Fab3 of omalizumab has higher efficacy than wild-type Fab of omalizumab when administered therapeutically in a 72-hour model of PCA.

A 25 μg/mL solution of human anti-DNP IgE was prepared by adding 7.5 μL of 6.68 mg/mL stock solution to 1992.5 μL of PBS. An injection of 20 μl of this solution would correspond to a dose of 500 ng of IgE. Animals (Tg hIgER mice) had their flanks shaved and then given an i/d injection into each flank at 2 pm on day 0. 20 μl of PBS was injected into the left flank of each animal as a negative control. Anti-DNP IgE (20 μl) was injected into the right flank. A total of 40 mice were injected. Administration of either wild-type omalizumab Fab or omalizumab Fab3 (with mutations S81R, Q83R, L158P [S77R, Q79R, L154P according to Kabat numbering]) was started 18 hours after IgE administration (8 am). Mice in two groups (n=8/group) received a dose of 100 mg/kg SC of either wild-type omalizumab Fab or omalizumab Fab3. 8 mice in the other group received SC PBS. Mice were reintroduced to the indicated doses 10 hours later (6 p.m.). At this time point, 28 hours after IgE administration, animals in two additional groups (n=8/group) were administered either wild-type omalizumab Fab or omalizumab Fab3 subcutaneously at a dose of 100 mg/kg. Animals in all groups were re-dosed at 8 a.m., 6 p.m., and again at 8 a.m. on the final day of the experiment. 72 hours after i.d. dosing (2 p.m.), all animals were given an IV injection of 100 μl of a solution of 1 mg/ml DNP-HSA, 2.5% w/v Evans blue dye prepared in 100 IU/ml heparin. After 1 hour, the animals were sacrificed in accordance with method 1. The skin from the sides around the i/d injection area was removed and a puncture biopsy was performed. Skin samples were placed in 700 μl of formamide and digested overnight at 55°C. After digestion, 100 µl x 2 liquid from each sample was withdrawn and placed into a 96-well ELISA plate. Absorbance was measured at 620 nm.

Conclusion:

The findings indicate that a mutant Fab form of omalizumab, which is capable of accelerating the dissociation of IgE from the high-affinity IgE receptor, FcεRI, is also able to reduce passive cutaneous anaphylaxis (as evidenced by inhibition of Evans blue dye leakage from the reaction site) to a statistically significant extent when compared with Fab omalizumab wild type. Mutations in the light chain that make this possible include, but are not necessarily limited to, S81R, Q83R and L158P with reference to SEQ ID NO:24, and the result is SEQ ID NO:39.

Example 5: Molecular Dynamics Simulation of Omalizumab Fab3 Complexed with IgE-Fc

Method:

The crystal structure of Fab3 of omalizumab complexed with the IgE Fc region (see Example 1) was obtained using the Molecular Operating Environment (MOE) 2014.0901 program (1), replacing missing side chains on some residues and missing loops between the Cε2 and Cε3 domains before performing molecular dynamics (MD) simulations using Amber 14 (2). The complex structure was supplemented with hydrogen and solvation was performed using the explicit water model TIP3P with 0.15 M dissolved NaCl salt in a truncated octahedral box in which the distance from each box face to the protein atoms was 10 Å. The system was tuned using Amber ff12SB force fields and GLYCAM_06j-1 oligosaccharides (3) and minimized by coupling a gradient algorithm for 50,000 steps with a 10.0 Å cutoff set for Coulomb and van der Waals interactions and a lattice-based neighbor list . The system was then gradually heated from 0 to 300 K in 125 ps at constant volume, followed by a 2.25 ns equilibration in the NPT ensemble constrained to all dissolved heavy atoms (the harmonic force constraint is 5.0). For electrostatics, we used fourth-order PME with a cutoff of 8.0 Å for Coulomb interactions with default Fourier distance and tolerance settings. Temperature was controlled with a weak interaction algorithm applied to the protein and solvent, respectively, with a time constant of 1.0 ps, and pressure was controlled using an isotropic Berendsen barostat applied to the entire system, with a time constant of 1.0 ps and compressibility of 4.46× 10 ^-5 bar ^-1 . Finally, a 1000 ns creation simulation was run without any constraints using the same parameters as for equilibration. To enable a 4-fs time step on the GPU infrastructure, the mass of protein and sugar hydrogen atoms was redistributed to 3.024 Da using ParmEd (4), while the mass of the atoms they are associated with was adjusted by the amount necessary for the total mass remained unchanged. The Fab structure of wild-type omalizumab in complex with the IgE Fc region was modeled based on the crystal structure of the mutant3 omalizumab complex with IgE-Fc by virtual mutation of R81 and R83 in the antibody light chain to wild-type serine and asparagine residues, respectively. MD simulations were performed using the same setup protocol as for mutant3.

The AmberTools cpptraj module (7) was used for cluster analysis of MD trajectories for both omalizumab Fab3 and omalizumab Fab in complex with IgE-Fc. A hierarchical agglomeration algorithm was used with an average bond distance between all clusters of less than 2.0 Å. The distance between the frameworks was calculated using the RMSD of the most accurately selected coordinates between the Cα atoms in the residues of the V region of the antibody light chain. Clustering was performed on only every 10th scaffold, and all other scaffolds were added to the clusters based on how close they were to the cluster centroids after clustering.

Bibliography

1. Molecular Operating Environment (MOE), 2014.09; Chemical Computing Group Inc., 1010 Sherbooke St. West, Suite #910, Montreal, QC, Canada, H3A 2R7, 2015.

2. Case, D. A., et al. AMBER 2014, 2014, University of California, San Francisco.

3. Kirschner, K. et al. GLYCAM06: A generalizable biomolecular force field. Carbohydrates. J. Comput. Chem., 2008, 29, 622-655.

4. http://parmed.github.io/ParmEd/html/index.html.

Results:

To examine how the S81R and Q83R mutations in the light chain of omalizumab Fab3 affect interaction with IgE, molecular dynamics simulations were performed at one-microsecond intervals for the omalizumab Fab and omalizumab Fab3 complexed with IgE-Fc structures, respectively. Trajectory snapshots were clustered and the 2 structures with the most populated cluster centers were analyzed, and in both clusters it was clearly seen that the two mutant arginine residues in mutant3 formed intense hydrogen bonding networks with residues D278 and S280 in the adjacent Cε2 domain of IgE. respectively. Visual inspection of the trajectory confirmed that the pairwise interactions of R81-S280 and R83-D278 are highly conserved and stable during the simulation, consequently, the unbent conformations of Cε2 relative to the closer Cε3 domain, as seen in the crystal structure, are captured by bridging with the antibody . Interestingly, although the double mutant arginine residues are spatially located near D278 and S280 of Cε2 in the omalizumab Fab3 crystal structure, they are not directly hydrogen bonded, as suggested by MD simulations. In the case of the omalizumab Fab, as expected, the hydrogen bond networks observed in the case of mutant3 are absent in the top 2 cluster center structures. Visual inspection of the trajectory shows that the Cε2 domains become less anchored to the antibody light chain scaffold and thus their positions relative to the closer Cε3 domain become more variable than in the case of omalizumab Fab3. In addition, the contact surface between Fab3 of omalizumab and IgE-Fc in their complex was subjected to IOTA analysis (IOTA is a tool for assessing statistical power to determine the probability of contact of a particular kind of atom at a protein contact surface or binding site), which predicted that positions S56, S64 and S71 of the light chain in Fab3 of omalizumab (referred to as SEQ ID NO:113) can be mutated to increase the affinity of omalizumab for IgE-Fc and will therefore increase the effect predicted for the S81R and Q38R mutations detailed above.

Conclusion:

In summary, based on the results of MD simulations, it can be hypothesized that the S81R and Q38R mutations promote the localization of IgE Cε2 domains on the antibody through direct electrostatic interactions and hydrogen bonds with the neighboring D278 and S280 residues in Cε2, and affect IgE-Fc plasticity by locking the extended conformation of Cε2. In addition, statistical methods predict that the S64M mutation, as well as S56 (change to D, E, Q or R) and S71 (change to D, E or M) will have a similar effect.

Example 6: Measuring the affinity of anti-IgE Fab for IgE-Fc using Biacore.

Biacore technology allows the measurement of interactions between biological molecules without the need for labeling. One of the interacting components, called the ligand, is either directly immobilized or trapped on the sensor surface, while the other, called the analyte, flows in solution above the retention surface. The sensor detects the change in mass on the sensor surface when the analyte binds to the ligand and when the analyte dissociates from the ligand. This corresponds to the processes of association and dissociation. In the kinetic assay, the anti-IgE Fab is the ligand captured on the sensor surface. IgE-Fc is an analyte and is captured by anti-IgE Fab. The association of IgE-Fc with the captured anti-IgE Fab and dissociation from it is controlled either by the flow of IgE-Fc over the sensor surface (association phase) or by the flow of buffer over the sensor surface (dissociation phase). Method details:

Device: Biacore 3000, GE Healthcare AB, Uppsala, Sweden

Touch chip: CM5. Catalog number BR100399

BIAnormalising solution: 70% (by weight) glycerin solution. Part of the BIAmaintenance suite. Catalog number BR100651. The BIAmaintenance kit was stored at 4°C.

Amino Bonding Kit: Catalog number BR100633. Ethyl 3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) was adjusted to 75 mg/mL in distilled water and stored in 200-μL aliquots at −70°C. N-Hydroxysuccinimide (NHS) was adjusted to a concentration of 11.5 mg/mL in distilled water and stored in 200-μL aliquots at −70°C. Ethanolamine hydrochloride-NaOH, pH 8.5, was stored at 4°C.

Buffers: The running buffer is HBS-EP (namely, 10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% P20 surfactant, reconstituted from 10x stock solution): catalog number BR100669. The immobilization buffer is acetate 4.0 (namely, 10 mM sodium acetate solution, pH 4.0). Catalog number BR100349. The buffer was stored at 4°C.

Ligand: The ligand was anti-IgE Fab molecules. They were transiently expressed as recombinant protein in HEK-293 cells and used without further purification.

Analyte: IgE-Fc, which is the Fc fragment of human IgE expressed as a recombinant protein in CHO cells and purified. Wild-type human IgE-Fc (domains Cε2-Cε4 numbered V224-K547 according to Dorrington & Bennich (1978) Immunol. Rev. 41:3-25) carrying the C225A mutation (SEQ ID NO:108) was used.

A Fab ₂ fragment of a goat anti-human IgG1 antibody (specific to a Fab ₂ fragment) (Jackson Immunolabs, catalog number 109-006-097) was immobilized on the sensor surface using standard amino group binding methods. Anti-IgE Fab was diluted in running buffer (HBS-EP), and approximately 200 response units were captured on the surface. IgE-Fc was diluted in running buffer, and serially diluted solutions with concentrations ranging from 2 nM to 125 pM were passed over the captured anti-IgE Fab. The association phase lasted 180 seconds, and the dissociation phase lasted 300 seconds. The sensor surface was regenerated with a 60-second exposure to 40 mM HCl, followed by a 60-second exposure to 10 mM NaOH, and then another 60-second exposure to 40 mM HCl. All binding data were processed using duplicate references using standard methods using the BiaEvaluation program.

Sample k _a (1/ms) x 10 ⁶ k _d (1/s) x 10 ^-3 K _D (nM) Omalizumab Fab (wild type) 1.97±0.021 2.10±0.005 1.07 Fab1 of omalizumab 2.17±0.021 1.95±0.005 0.902 Fab2 of omalizumab 2.21±0.029 2.91±0.007 1.32 Fab3 omalizumab 2.36±0.021 1.78±0.005 0.754

Table 5: Omalizumab Fab and mutant affinities for IgE-Fc as measured in the Biacore kinetic assay. The dissociation constant (K _D ) is calculated as k _d /k _a .

Sample k _a (x10 ⁶ ) 1/ms k _d (x10 ^-3 ) 1/s K _D (pm) DT 2.86 1.42 496 S56D 3.26 0.713 219 S56E 2.86 0.717 251 S71E 2.57 0.583 226 S71D 2.51 0.696 278

Table 6: Omalizumab (WT) Fab and mutant affinities for IgE-Fc as measured in the Biacore kinetic assay. The dissociation constant (K _D ) is calculated as k _d /k _a . Four mutations are found in the original wild-type sequence (light chain, SEQ ID NO:20).

Sample k _a (x10 ⁶ ) 1/ms k _d (x10 ^-3 ) 1/s K _D (pm) DT 2.25 16.2 721 S71E 1.7 6.25 368 S56D 2.17 7.09 327 S71D, S56D 2.13 8.11 380 S71E, S56D 1.85 8.17 443 S71M, S56D 2.32 5.45 238 S71D, S56E 1.96 8.33 426 S71E, S56E 1.94 6.66 343 S71M, S56E 1.76 4.54 258 S71D, S56Q 1.85 6.12 331 S71E, S56Q 1.87 5.03 268 S71M, S56Q 1.44 4.11 286 S71D, S56R 1.5 4.86 325 S71E, S56R 1.65 4.63 281 S71M, S56R 1.05 5.58 534

Table 7a: Omalizumab Fab (WT) and mutant affinities for IgE-Fc as measured in the Biacore kinetic assay. The dissociation constant (K _D ) is calculated as k _d /k _a . Mutations are indicated by reference to the light chain, SEQ ID NO:20.

VL SEQ ID NO: Sample k _a (1/ms) k _d (1/s) _KD (M) K _D (pm) 20 Fab omalizumab DT 2.52E+06 2.38E-03 9.42E-10 942 132 S64M_S81R_Q83R_S56D_S71M 3.23E+06 9.79E-04 3.04E-10 304 139 S64K_S81R_Q83R_S56D_S71M 2.85E+06 1.02E-03 3.58E-10 358 145 S64Q_S81R_Q83R_S56D_S71M 2.99E+06 9.12E-04 3.05E-10 305 146 S64R_S81R_Q83R_S56D_D71M 2.67E+06 6.27E-04 2.35E-10 235 158 S64M_S81R_Q83R_D56D_D71M_S67W_S80N 3.19E+06 5.40E-04 1.69E-10 169 159 S64M_S81R_Q83R_D56D_D71M_S67Y_S80N 3.07E+06 4.69E-04 1.53E-10 153

Table 7b: Omalizumab Fab affinity for IgE-Fc as measured in the Biacore kinetic assay. The dissociation constant (K _D ) is calculated as k _d /k _a . The mutations are in the light chain (referred to as SEQ ID NO:20). The light chain variable region sequences of the Fab mutants are indicated in the left column (VL SEQ ID NO:).

Sample _{% H2O2} Period of time k _a (1/ms) k _d (1/s) _KD (M) K _D (pm) S64M_S81R_Q83R_S56D_S71M
(SEQ ID NO:132) initially 3.70E+06 1.18E-03 3.2E-10 320 0.00% 4 hours 3.43E+06 1.38E-03 4.03E-10 403 1 day 3.40E+06 1.46E-03 4.3E-10 430 3 days 3.37E+06 1.38E-03 4.09E-10 409 7 days 3.25E+06 1.36E-03 4.18E-10 418 14 days 3.08E+06 1.22E-03 3.97E-10 397 0.1 4 hours 3.46E+06 1.44E-03 4.18E-10 418 1 day 3.41E+06 1.26E-03 3.71E-10 371 3 days 3.49E+06 1.23E-03 3.53E-10 353 7 days 5.64E+06 1.36E-03 2.41E-10 241 14 days 3.64E+06 1.20E-03 3.31E-10 331

Table 7c: Omalizumab Fab mutant affinity for IgE-Fc as measured in the Biacore kinetic assay. The dissociation constant (K _D ) is calculated as k _d /k _a . The mutations are in the light chain (referred to as SEQ ID NO:20). Samples were tested under enhanced oxidation conditions, and methionine oxidation levels at each time point were determined by mass spectrometry.

Conclusion:

The findings indicate that omalizumab Fab mutations may result in increased omalizumab Fab affinity for IgE-Fc. The best combination of mutations from the point of view of increasing affinity is the combination of S71M with S56D. This increase in affinity is mainly caused by a decrease in the rate of Fab dissociation from IgE-Fc. Affinity of Fab1 of omalizumab (with mutations S81R, Q83R [S77R, Q79R according to Kabat numbering]) and Fab3 (with mutations S81R, Q83R, L158P [S77R, Q79R, L154P according to Kabat numbering] with reference to SEQ ID NO:125 ) is also increased, due to improved interaction of the antibody with Cε2 IgE-Fc compared to non-mutant omalizumab Fab.

Example 7: Measurement of accelerated dissociation of IgE-Fc from immobilized sFcεRIα using Biacore.

The effect of anti-IgE Fab on the dissociation of IgE-Fc from sFcεRIα was determined by the methods described in Example 2 (assay method 2). All anti-IgE Fabs were expressed in HEK-293 cells, purified by standard methods, and quantified by absorbance at 280 nm using calculated molar extinction coefficients. In this assay, the IgE-Fc concentration was 2 nM and the dissociation time was 200 seconds. The amount of IgE-Fc dissociated from immobilized sFcεRIα was calculated as a function of the initially bound amount, and the dissociation rate was calculated as the amount of IgE-Fc dissociated from immobilized sFcεRIα normalized to the initially bound amount as a function of time elapsed.

S80N (pdb numbering) is thought to interact with D278 in Cε2 IgE and S67W/Y (pdb numbering) interacts with T298 in Cε2 IgE.

Sample Mutations Captured IgE-Fc, EO IgE-Fc after dissociation, EO % Dissociation Apparent dissociation rate (1/s) Fab3 omalizumab S81R Q83R L158P 152.1 125.9 17.0 6.25E-04 sample 1 S64M S81R Q83R L158P 56D 71M 135.3 71.0 47.5 2.15E-03 sample 2 S64M S81R Q83R L158P 56E 71M 120.5 71.6 40.6 1.74E-03 sample 3 S64M S81R Q83R L158P 56Q 71M 97.5 65.4 33.0 1.33E-03 sample 4 S64M S81R Q83R L158P 56Q 71E 87.6 62.1 29.1 1.15E-03 sample 5 S64M S81R Q83R L158P 56R 71E 80.0 64.4 19.5 7.24E-04 sample 6 S64M S81R Q83R 56D 71M 85.9 50.9 40.7 1.74E-03 sample 7 S64M S81R Q83R 56E 71M 101.8 66.6 34.6 1.42E-03 sample 8 S64M S81R Q83R 56Q 71M 109.1 77.6 28.9 1.14E-03 sample 9 S64M S81R Q83R 56Q 71E 107.8 78.0 27.6 1.08E-03 sample 10 S64M S81R Q83R 56R 71E 102.8 84.3 18.1 6.64E-04 sample 11 S56D S64M S71E S81R Q83R 156.7 110.5 29.5 1.17E-03 sample 12 S56D S64M S71E S81R Q83R S67W S80N 155.1 92.0 40.7 1.74E-03 sample 13 S56D S64M S71E S81R Q83R S67Y S80N 137.6 71.5 48.0 2.18E-03 control, buffer 126.3 116.6 7.8 2.70E-04

Table 8: Calculation of the amount of IgE-Fc dissociated from immobilized sFcεRIα. The initial binding of IgE to sFcεRIα was set to 100%, and the dissociation was calculated relative to this. The apparent dissociation rate is based on an assumed single-rate model.

Conclusion:

The findings demonstrate that a mutant Fab form of omalizumab can accelerate the dissociation of IgE from an immobilized form of the high-affinity IgE receptor, FcεRI. Mutations in the light chain (SEQ ID NO:20) that make this possible include, but are not necessarily limited to, mutations at positions S56, S64, S67, S71, S80, S81, Q83 and L158 (S52, S60, S63, S67 , S76, S77, Q79 and L154, respectively, according to Kabat numbering).

Example 8: Enhanced Oxidation of Fab Mutant Omalizumab

Anti-IgE Fab samples were subjected to an enhanced oxidation regimen to determine the effect of oxidation of methionine residues in the light chain variable region on the Fab's affinity for IgE-Fc and on the ability to accelerate dissociation of the IgE-Fc:sFcεRIα complex. The omalizumab mutant Fab was incubated with 0.1% and 1% (v/v) hydrogen peroxide solution for up to 14 days at room temperature. After incubation, the sample buffer was changed back to PBS, pH 7.4, and the concentration was determined by absorbance at 280 nm using the calculated extinction coefficient. Mass spectral analysis was performed to determine the amount of oxidized methionine residues in the light chain variable regions by reduction and alkylation of the material under denaturing conditions, followed by trypsin digestion (50 μg/ml trypsin for 180 minutes at 37°C, followed by TFA quenching), and then LC-MS analysis on a Thermo Orbitrap Q Exactive Plus mass spectrometer. The percentage of oxidized methionine residues was calculated based on the assumption that no oxidized methionine residues were present during synthesis and compared with reference material, which was stored at 4°C.

% Oxidized methionine residues present at each time point IgE control 4 hours 1 day 3 days 7 days 14 days IgE control M37 (LC) 0.30% 0.40% 0.30% 0.40% 0.60% 0.90% 0.30% M64 (LC) 18.10% 81.70% 99.70% 99.90% 99.90% 99.90% 20.00% M71 (LC) 6.70% 90.00% 99.70% 99.80% 99.80% 99.90% 6.90%

Table 9: Percentage of oxidized methionine residues in the light chain variable region of the omalizumab mutant Fab. This omalizumab Fab mutant has the following mutations: S64M_S81R_Q83R_S56D_S71M (referred to as SEQ ID NO:20).

Conclusion:

The data obtained show that it is possible to obtain mutant Fab omalizumab, in which there is almost complete oxidation of critical light chain methionine residues (M64 and M71). From these data, material from day 1, day 3, and day 7 samples was pooled and used to determine the effect of methionine oxidation on the affinity of the omalizumab mutant Fab for IgE-Fc and on the ability to accelerate dissociation of the IgE-Fc:sFcεRIα complex.

Example 9: Measuring Accelerated Dissociation of IgE-Fc from sFcεRIα Using FRET

The effect of anti-IgE Fab on the dissociation of IgE-Fc from sFcεRIα was determined in a homogeneous FRET assay. All anti-IgE Fabs were expressed in HEK-293 cells, purified by standard methods, and quantified by absorbance at 280 nm using calculated molar extinction coefficients. The FRET assay used Tb-labeled IgE-Fc as the donor and Alexa 488-labeled sFcεRIα as the acceptor. Both reagents were mixed and equilibrated at room temperature for 60 minutes with a final assay concentration of 1 nM. Anti-IgE Fab was added to the mixture to a final assay concentration of 500 nM and fluorescence readings (excitation at 330 nm, emission at 495 and 520 nm) were taken every 20 minutes for 800 minutes. Fluorescence emission was plotted against time, and the rate of dissociation of IgE-Fc from sFcεRIα was calculated as the half-life of the complex. The obtained data are presented in Tables 10 and 11.

VL SEQ ID NO: Sample Half-life of the complex (min) 20 Fab omalizumab DT 95.34 132 S64M_S81R_Q83R_S56D_S71M 22.94 145 S64K_S81R_Q83R_S56D_S71M 29.54 146 S64Q_S81R_Q83R_S56D_S71M 28.1 139 S64R_S81R_Q83R_S56D_S71M 19.26 158 S64M_S81R_Q83R_S56D_S71M_S67W_S80N 17.02 159 S64M_S81R_Q83R_S56D_S71M_S67Y_S80N 17

Table 10: Effect of anti-IgE Fab (WT and mutants) on the dissociation of IgE-Fc from sFcεRIα in a homogeneous FRET assay. Dissociation of the IgE-Fc:sFcεRIα complex is a two-phase disintegration. The fast initial phase is the phase in which most of the IgE-Fc:sFcεRIα complex dissociates and from which the half-life of the complex is determined. The mutations are in the light chain (referred to as SEQ ID NO:20). The light chain variable region sequences of the Fab mutants are indicated in the left column (VL SEQ ID NO:).

Sample Half-life of the complex (min) buffer 1240 S64M_S81R_Q83R_S56D_S71M 10.8 S64M_S81R_Q83R_S56D_Q71M, enhanced oxidation 17.3

Table 11: Effect of methionine oxidation on the ability of anti-IgE Fab to cause dissociation of IgE-Fc from sFcεRIα in a homogeneous FRET assay. Dissociation of the IgE-Fc:sFcεRIα complex is a two-phase disintegration. The fast initial phase is the phase in which most of the IgE-Fc:sFcεRIα complex dissociates and from which the half-life of the complex is determined.

Conclusions:

The findings indicate that mutant forms of omalizumab are capable of causing dissociation of IgE-Fc from sFcεRIα at a faster rate than the wild-type sequence. In particular, mutations in the light chain (with reference to SEQ ID NO:20) that make this possible include, but are not necessarily limited to, mutations at positions S64 and S67. In addition, oxidation of methionine residues (M64 and M71, SEQ ID NO:20) does not have a significant effect on the ability of Fab to accelerate the dissociation of the IgE-Fc:sFcεRIα complex.

This specification describes embodiments of the invention. However, one skilled in the art will appreciate that various modifications and changes may be made without departing from the spirit of the invention as set forth in the following claims. Accordingly, the specification is to be considered as illustrative and not as limiting of the invention, and all such modifications are intended to be within the scope of the invention.

Sequences

heavy chain

SEQ ID NO:1

v-region

evqlvesggg lvqpggslrl scavsgysit sgyswnwirq apgkglewva sitydgstny npsvkgriti srddskntfy lqmnslraed tavyycargs hyfghwhfav wgqgtlvtvs s

SEQ ID NO:2

v-region

t gaagggccg catcaccatt tcgcgcgacg acagcaagaa tactttttac ctccaaatga acagcctgcg ggccgaagat actgccgtgt actactgcgc gcggggatca cattacttcg ggcactggca cttcgccgtc tggggacagg gcaccctcgt cactgtctcg agc

SEQ ID NO:3

v-region with signal sequence, which is underlined and italicized

mkwvtfisll flfssaysev qlvesggglv qpggslrlsc avsgysitsg yswnwirqap gkglewvasi tydgstnynp svkgritisr ddskntfylq mnslraEDTA vyycargshy fghwhfavwg qgtlvtvss

SEQ ID NO:4

v-region with signal sequence, which is underlined and italicized

atgaagtggg tcaccttcat ctccctgctg tttctgttct ccagcgccta ctccgaagtg cagttggtgg agtcgggtgg agggctggtg cagcctggcg gtagcctgag gctgtcctgt gccgtgtccg gatactccat tacctccggc tactcgtgga actggatcag acaggctccc ggaa agggac ttgagtgggt ggcgtccatc acctacgacg gctcaaccaa ctataacccg tccgtgaagg gccgcatcac catttcgcgc gacgacagca agaatacttt ttacctccaa atgaacagcc tgcgggccga agatactgcc gtgtactact gcgcgcgggg atcacattac ttcgggcact ggcact tcgc cgtctgggga cagggcaccc tcgtcactgt ctcgagc

SEQ ID NO:5

v-region+CH1 constant region gamma-1

evqlvesggg lvqpggslrl scavsgysit sgyswnwirq apgkglewva sitydgstny npsvkgriti srddskntfy lqmnslraed tavyycargs hyfghwhfav wgqgtlvtvs sastkgpsvf plapssksts ggtaalgclv kdyfpepvtv swnsgaltsg vht fpavlqs sglyslssvv tvpssslgtq tyicnvnhkp sntkvdkkve pksc

SEQ ID NO:6

v-region+CH1 constant region gamma-1

t gaagggccg catcaccatt tcgcgcgacg acagcaagaa tactttttac ctccaaatga acagcctgcg ggccgaagat actgccgtgt actactgcgc gcggggatca cattacttcg ggcactggca cttcgccgtc tggggacagg gcaccctcgt cactgtctcg agcgcttcta caaagggccc atc ggtcttc cccctggcac cctcctccaa gagcacctct gggggcacag cggccctggg ctgcctggtc aaggactact tccccgaacc ggtgacggtg tcgtggaact caggcgccct gaccagcggc gtgcacacct tcccggctgt cctacagtcc tcaggactct actccctcag cagcgtggtg accgtgccct ccagcagctt gggcacccag acctacatct gcaacgtgaa tcacaagccc agcaacacca aggtggacaa gaaagttgag cccaaatctt gt

SEQ ID NO:7

v-region+CH1 of gamma-1 constant region with signal sequence which is underlined and italicized

mkwvtfisll flfssaysev qlvesggglv qpggslrlsc avsgysitsg yswnwirqap gkglewvasi tydgstnynp svkgritisr ddskntfylq mnslraEDTA vyycargshy fghwhfavwg qgtlvtvssa stkgpsvfpl apsskstsgg taalgclvkd y fpepvtvsw nsgaltsgvh tfpavlqssg lyslssvvtv pssslgtqty icnvnhkpsn tkvdkkvepk sc

SEQ ID NO:8

v-region+CH1 of gamma-1 constant region with signal sequence which is underlined and italicized

atgaagtggg tcaccttcat ctccctgctg tttctgttct ccagcgccta ctccgaagtg cagttggtgg agtcgggtgg agggctggtg cagcctggcg gtagcctgag gctgtcctgt gccgtgtccg gatactccat tacctccggc tactcgtgga actggatcag acaggctccc ggaa agggac ttgagtgggt ggcgtccatc acctacgacg gctcaaccaa ctataacccg tccgtgaagg gccgcatcac catttcgcgc gacgacagca agaatacttt ttacctccaa atgaacagcc tgcgggccga agatactgcc gtgtactact gcgcgcgggg atcacattac ttcgggcact ggcact tcgc cgtctgggga cagggcaccc tcgtcactgt ctcgagcgct tctacaaagg gcccatcggt cttccccctg gcaccctcct ccaagagcac ctctgggggc acagcggccc tgggctgcct ggtcaaggac tacttccccg aaccggtgac ggtgtcgtgg aactcaggcg ccctgaccag cggcgtgcac accttcccgg ctgtcctaca gtcctcagga ctctactccc tcagcagcgt ggtgaccgtg ccctccagca gcttgggcac ccagacctac atctgcaacg tgaatcacaa gc ccagcaac accaaggtgg acaagaaagt tgagcccaaa tcttgt

SEQ ID NO:9

v-region+full-length gamma-1 constant region

evqlvesggg lvqpggslrl scavsgysit sgyswnwirq apgkglewva sitydgstny npsvkgriti srddskntfy lqmnslraed tavyycargs hyfghwhfav wgqgtlvtvs sastkgpsvf plapssksts ggtaalgclv kdyfpepvtv swnsgaltsg vht fpavlqs sglyslssvv tvpssslgtq tyicnvnhkp sntkvdkkve pkscdkthtc ppcpapellg gpsvflfppk pkdtlmisrt pevtcvvvdv shedpevkfn wyvdgvevhn aktkpreeqy nstyrvvsvl tvlhqdwlng keykckvsnk al papiekti skakgqprep qvytlppsrE eMtknqvslt clvkgfypsd iavewesngq pennykttpp vldsdgsffl yskltvdksr wqqgnvfscs vmhealhnhy tqkslslspg k

SEQ ID NO:10

v-region+full-length gamma-1 constant region

t gaagggccg catcaccatt tcgcgcgacg acagcaagaa tactttttac ctccaaatga acagcctgcg ggccgaagat actgccgtgt actactgcgc gcggggatca cattacttcg ggcactggca cttcgccgtc tggggacagg gcaccctcgt cactgtctcg agcgcttcta caaagggccc ct ccgtgttc ccgctcgctc catcatcgaa gtctaccagc ggaggcactg cggctctcgg ttgcctcgtg aaggactact tcccggagcc ggtgaccgtg tcgtggaaca gcggagccct gaccagcggg gtgcacacct ttccggccgt cttgcagtca agcggccttt actccctgtc atcagtggtg actgtcccgt ccagctcatt gggaacccaa acctacatct gcaatgtgaa tcacaaacct agcaacacca aggttgacaa gaaagtcgag cccaaatcgt gtgacaagac tcacact tgt ccgccgtgcc cggcacccga actgctggga ggtcccagcg tctttctgtt ccctccaaag ccgaaagaca cgctgatgat ctcccgcacc ccggaggtca cttgcgtggt cgtggacgtg tcacatgagg acccagaggt gaagttcaat tggtacgtgg atggcgtcga agtccacaat gccaaaacta agcccagaga agaacagtac aattcgacct accgcgtcgt gtccgtgctc acggtgttgc atcaggattg gctgaacggg aaggaataca agtgcaaagt gtccaacaag gcgctgccgg caccgatcga gaaaactatc tccaaagcga agggacagcc tagggaacct caagtctaca cgctgccacc atcacgggaA gaaAtgacta agaatcaagt ctcactgact tgtctggtga aggggtttta ccctagcgac attgccgtgg agtgggaatc caacggccag ccagagaaca actacaagac tacccctcca gt gctcgact cggatggatc gttcttcctt tactcgaagc tcaccgtgga taagtcccgg tggcagcagg gaaacgtgtt ctcctgctcg gtgatgcatg aagccctcca taaccactat acccaaaagt cgctgtccct gtcgccggga aag

SEQ ID NO:11

v-region + full-length gamma-1 constant region with signal sequence underlined and italicized

mkwvtfisll flfssaysev qlvesggglv qpggslrlsc avsgysitsg yswnwirqap gkglewvasi tydgstnynp svkgritisr ddskntfylq mnslraEDTA vyycargshy fghwhfavwg qgtlvtvssa stkgpsvfpl apsskstsgg taalgclvkd y fpepvtvsw nsgaltsgvh tfpavlqssg lyslssvvtv pssslgtqty icnvnhkpsn tkvdkkvepk scdkthtcpp cpapellggp svflfppkpk dtlmisrtpe vtcvvvdvsh edpevkfnwy vdgvevhnak tkpreeqyns tyrv vsvltv lhqdwlngke ykckvsnkal papiektisk akgqprepqv ytlppsrEeM tknqvsltcl vkgfypsdia vewesngqpe nnykttppvl dsdgsfflys kltvdksrwq qgnvfscsvm healhnhytq kslslspgk

SEQ ID NO:12

v-region + full-length gamma-1 constant region with signal sequence underlined and italicized

atgaagtggg tcaccttcat ctccctgctg tttctgttct ccagcgccta ctccgaagtg cagttggtgg agtcgggtgg agggctggtg cagcctggcg gtagcctgag gctgtcctgt gccgtgtccg gatactccat tacctccggc tactcgtgga actggatcag acaggctccc ggaa agggac ttgagtgggt ggcgtccatc acctacgacg gctcaaccaa ctataacccg tccgtgaagg gccgcatcac catttcgcgc gacgacagca agaatacttt ttacctccaa atgaacagcc tgcgggccga agatactgcc gtgtactact gcgcgcgggg atcacattac ttcgggcact ggcact tcgc cgtctgggga cagggcaccc tcgtcactgt ctcgagcgct tctacaaagg gcccctccgt gttcccgctc gctccatcat cgaagtctac cagcggaggc actgcggctc tcggttgcct cgtgaaggac tacttcccgg agccggtgac cgtgtcgtgg aacagcggag ccctgaccag cggggtgcac acctttccgg ccgtcttgca gtcaagcggc ctttactccc tgtcatcagt ggtgactgtc ccgtccagct cattgggaac ccaaacctac atctgcaatg tgaatcacaa acc tagcaac accaaggttg acaagaaagt cgagcccaaa tcgtgtgaca agactcacac ttgtccgccg tgcccggcac ccgaactgct gggaggtccc agcgtctttc tgttccctcc aaagccgaaa gacacgctga tgatctcccg caccccggag gtcacttgcg tggtcgtgga cgt gtcacat gaggacccag aggtgaagtt caattggtac gtggatggcg tcgaagtcca caatgccaaa actaagccca gagaagaaca gtacaattcg acctaccgcg tcgtgtccgt gctcacggtg ttgcatcagg attggctgaa cgggaaggaa tacaagtgca aagtgtccaa caaggcgctg ccggcaccga tcgagaaaac tatctccaaa gcgaagggac agcctaggga acctcaagtc tacacgctgc caccatcacg ggaAgaaAtg actaagaatc aagtctcact gacttgtctg gtgaaggggt tttaccctag cga cattgcc gtggagtggg aatccaacgg ccagccagag aacaactaca agactacccc tccagtgctc gactcggatg gatcgttctt cctttactcg aagctcaccg tggataagtc ccggtggcag cagggaaacg tgttctcctg ctcggtgatg catgaagccc tccataacca ctatacccaa aagtcgctg t ccctgtcgcc gggaaag

SEQ ID NO:13

FR H1

evqlvesggg lvqpggslrl scavs

SEQ ID NO:14

CDRH1

gysitsgysw n

SEQ ID NO:15

FR H2

wirqapgkgl ewva

SEQ ID NO:16

CDRH2

citydgstny npsvkg

SEQ ID NO:17

FR H3

ritisrddsk ntfylqmnsl raEDTAvyyc ar

SEQ ID NO:18

CDRH3

gshyfghwhf av

SEQ ID NO:19

FR H4

wgqgtlvtvs

LIGHT CHAIN

SEQ ID NO:20

Omalizumab_v-region

DIQLTQSPSS LSASVGDRVT ITCRASQSVD YDGDSYMNWY QQKPGKAPKL LIYAASYLES GVPSRFSGSG SGTDFTLTIS SLQPEDFATY YCQQSHEDPY TFGQGTKVEI K

SEQ ID NO:21

Omalizumab_v-region

gatattcagc tgactcagag cccgagctca ctctccgctt ccgtggggga tagagtgacc atcacttgcc gggcatccca gtcggtggac tacgacggag actcctacat gaactggtat cagcagaagc ccggaaaagc cccaaagttg ctgatctacg ccgcctcata ccttgaaagc ggcgtgcctt cg cgcttctc gggaagcggg tcgggcaccg atttcaccct gaccatttcg tcgctgcagc cggaggactt cgcgacttac tactgccaac agtcccacga ggacccctat acgtttggcc agggaaccaa ggtcgaaatc aag

SEQ ID NO:22

Omalizumab_v-region with signal sequence, which is underlined and italicized

mkwvtfisll flfssaysdi qltqspssls asvgdrvtit crasqsvdyd gdsymnwyqq kpgkapklli yaasylesgv pSrfsgsgsg tdftltisSl Qpedfatyyc qqshedpytf gqgtkveik

SEQ ID NO:23

Omalizumab_v-region with signal sequence, which is underlined and italicized

atgaagtggg tcaccttcat ctccctgctg tttctgttct cctccgccta ctccgatatt cagctgactc agagcccgag ctcactctcc gcttccgtgg gggatagagt gaccatcact tgccgggcat cccagtcggt ggactacgac ggagactcct acatgaactg gtatcagcag aagcccggaa aagcccca aa gttgctgatc tacgccgcct cataccttga aagcggcgtg ccttcgcgct tctcgggaag cgggtcgggc accgatttca ccctgaccat ttcgtcgctg cagccggagg acttcgcgac ttactactgc caacagtccc acgaggaccc ctatacgttt ggccagggaa ccaaggtcga a atcaag

SEQ ID NO:24

Omalizumab_v-region+kappa constant region

DIQLTQSPSS LSASVGDRVT ITCRASQSVD YDGDSYMNWY QQKPGKAPKL LIYAASYLES GVPSRFSGSG SGTDFTLTIS SLQPEDFATY YCQQSHEDPY TFGQGTKVEI KRTVAAPSVF IFPPSDEQLK SGTASVVCLL NNFYPREAKV QWKVDNALQS GNSQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEV THQGLSSPVT KSFNRGEC

SEQ ID NO:25

Omalizumab_v-region+kappa constant region

gatattcagc tgactcagag cccgagctca ctctccgctt ccgtggggga tagagtgacc atcacttgcc gggcatccca gtcggtggac tacgacggag actcctacat gaactggtat cagcagaagc ccggaaaagc cccaaagttg ctgatctacg ccgcctcata ccttgaaagc ggcgtgcctt cg cgcttctc gggaagcggg tcgggcaccg atttcaccct gaccatttcg tcgctgcagc cggaggactt cgcgacttac tactgccaac agtcccacga ggacccctat acgtttggcc agggaaccaa ggtcgaaatc aagcgtacgg tagcggcccc atctgtcttc atcttcccgc catctgatga gca gttgaaa tctggaactg cctctgttgt gtgcctgctg aataacttct atcccagaga ggccaaagta cagtggaagg tggataacgc cctccaatcg ggtaactccc aggagagtgt cacagagcag gacagcaagg acagcaccta cagcctcagc agcaccctga cgctgagcaa agcagactac gagaaacaca aagtctacgc ctgcgaagtc acccatcagg gcctgagctc gcccgtcaca aagagcttca acaggggaga gtgt

SEQ ID NO:26

Omalizumab_v-region+kappa constant region with signal sequence underlined and italicized

mkwvtfisll flfssaysDI QLTQSPSSLS ASVGDRVTIT CRASQSVDYD GDSYMNWYQQ KPGKAPKLLI YAASYLESGV PSRFSGSGSG TDFTLTISSL QPEDFATYYC QQSHEDPYTF GQGTKVEIKR TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN SQ ESVTEQDS KDSTYSLSST LTLSKADYEK HKVYACEVTH QGLSSPVTKS FNRGEC

SEQ ID NO:27

Omalizumab_v-region+kappa constant region with signal sequence underlined and italicized

atgaagtggg tcaccttcat ctccctgctg tttctgttct cctccgccta ctccgatatt cagctgactc agagcccgag ctcactctcc gcttccgtgg gggatagagt gaccatcact tgccgggcat cccagtcggt ggactacgac ggagactcct acatgaactg gtatcagcag aagcccggaa aagcccca aa gttgctgatc tacgccgcct cataccttga aagcggcgtg cctTCgcgct tctcgggaag cgggtcgggc accgatttca ccctgaccat ttcgTCGctg CAgccggagg acttcgcgac ttactactgc caacagtccc acgaggaccc ctatacgttt ggccagggaa ccaaggtcga aatca agcgt acggtagcgg ccccatctgt cttcatcttc ccgccatctg atgagcagtt gaaatctgga actgcctctg ttgtgtgcct gctgaataac ttctatccca gagaggccaa agtacagtgg aaggtggata acgcccTCca atcgggtaac tcccaggaga gtgtcacaga gcaggacagc aagggacagca cctacagcct cagcagcacc ctgacgctga gcaaagcaga ctacgagaaa cacaaagtct acgcctgcga agtcacccat cagggcctga gctcgcccgt cacaaagagc ttcaac aggg gagagtgt

SEQ ID NO:28

FR L1

DIQLTQSPSS LSASVGDRVT ITC

SEQ ID NO:29

CDRL1

rasqsvdydg dsymn

SEQ ID NO:30

FR L2

WYQQKPGKAP KLLIY

SEQ ID NO:31

CDRL2

aasyles

SEQ ID NO:32

FR L3

GVPSRFSGSG SGTDFTLTIS SLQPEDFATY YC

SEQ ID NO:33

CDRL3

qqshedpyt

SEQ ID NO:34

FR L4

FGQGTKVEIK

MUTANT LIGHT CHAIN

SEQ ID NO:35

S60M_S77R_Q79R_v-area (Kabat)

DIQLTQSPSS LSASVGDRVT ITCRASQSVD YDGDSYMNWY QQKPGKAPKL LIYAASYLES GVPMRFSGSG SGTDFTLTIS RLRPEDFATY YCQQSHEDPY TFGQGTKVEI K

SEQ ID NO:36

S60M_S77R_Q79R_v-area (Kabat)

gatattcagc tgactcagag cccgagctca ctctccgctt ccgtggggga tagagtgacc atcacttgcc gggcatccca gtcggtggac tacgacggag actcctacat gaactggtat cagcagaagc ccggaaaagc cccaaagttg ctgatctacg ccgcctcata ccttgaaagc ggcgtgccta tgc gcttctc gggaagcggg tcgggcaccg atttcaccct gaccatttcg agactgaggc cggaggactt cgcgacttac tactgccaac agtcccacga ggacccctat acgtttggcc agggaaccaa ggtcgaaatc aag

SEQ ID NO:37

S60M_S77R_Q79R_v - region with signal sequence, which is underlined and italicized (Kabat)

mkwvtfisll flfssaysdi qltqspssls asvgdrvtit crasqsvdyd gdsymnwyqq kpgkapklli yaasylesgv pmrfsgsgsg tdftltisrl rpedfatyyc qqshedpytf gqgtkveik

SEQ ID NO:38

S60M_S77R_Q79R_v - region with signal sequence, which is underlined and italicized (Kabat)

ATGAAGTGGGGGGGGGGGGGGCTGCTGTGTGTGTCTTC CCTCCGCCTA CTCCGATTC CAGAGCCCCCCCCCCGAG CTCACTCTC GCTCCGTGG GGGGAGT GACCCAGTCACT TGCGCGCGCAT CCCAGGCAT CCCCAGGCAT CCCAGGCAT CCCAGGCAT CGGT GGACTACGAC GGAGAGAGACTCCTCCTG GTATCAGCAG AAGCCCGAA AAGCCCCCCAAA GTTGCTGCCCCCCCCCCCCCCCCGCGCGCGCGCGCGCGCGCGCGGGGGGCGCGGCGCGGCGCGGGCGCGGGCGCGGCGCGGGC GatttCA CCCTGACCAT TTCGAGAGAGAGGGGGGGGCGCGCGCGCTACGC CAACACAGGGAGGAGGAGGAGGGGGGAA CCAAGGGGAA Aatcaag

SEQ ID NO:39

S60M_S77R_Q79R_v-region+kappa constant region including L154P (Kabat)

DIQLTQSPSS LSASVGDRVT ITCRASQSVD YDGDSYMNWY QQKPGKAPKL LIYAASYLES GVPMRFSGSG SGTDFTLTIS RLRPEDFATY YCQQSHEDPY TFGQGTKVEI KRTVAAPSVF IFPPSDEQLK SGTASVVCLL NNFYPREAKV QWKVDNAPQS GNSQESVTEQ DSKDSTYSLS ST LTLSKADY EKHKVYACEV THQGLSSPVT KSFNRGEC

SEQ ID NO:40

S60M_S77R_Q79R_v-region+kappa constant region including L154P (Kabat)

gatattcagc tgactcagag cccgagctca ctctccgctt ccgtggggga tagagtgacc atcacttgcc gggcatccca gtcggtggac tacgacggag actcctacat gaactggtat cagcagaagc ccggaaaagc cccaaagttg ctgatctacg ccgcctcata ccttgaaagc ggcgtgccta tgc gcttctc gggaagcggg tcgggcaccg atttcaccct gaccatttcg agactgaggc cggaggactt cgcgacttac tactgccaac agtcccacga ggacccctat acgtttggcc agggaaccaa ggtcgaaatc aagcgtacgg tagcggcccc atctgtcttc atcttcccgc catctgatga gcagtt gaaa tctggaactg cctctgttgt gtgcctgctg aataacttct atcccagaga ggccaaagta cagtggaagg tggataacgc cccgcaatcg ggtaactccc aggagagtgt cacagagcag gacagcaagg acagcaccta cagcctcagc agcaccctga cgctgagcaa agcagactac gagaaacaca aagtctacgc ctgcgaagtc acccatcagg gcctgagctc gcccgtcaca aagagcttca acaggggaga gtgt

SEQ ID NO:41

S60M_S77R_Q79R_v region+kappa constant region including L154P, with signal sequence underlined and italicized (Kabat)

mkwvtfisll flfssaysDI QLTQSPSSLS ASVGDRVTIT CRASQSVDYD GDSYMNWYQQ KPGKAPKLLI YAASYLESGV PMRFSGSGSG TDFTLTISRL RPEDFATYYC QQSHEDPYTF GQGTKVEIKR TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNAPQSGN SQ ESVTEQDS KDSTYSLSST LTLSKADYEK HKVYACEVTH QGLSSPVTKS FNRGEC

SEQ ID NO:42

S60M_S77R_Q79R_v region+kappa constant region including L154P, with signal sequence underlined and italicized (Kabat)

atgaagtggg tcaccttcat ctccctgctg tttctgttct cctccgccta ctccgatatt cagctgactc agagcccgag ctcactctcc gcttccgtgg gggatagagt gaccatcact tgccgggcat cccagtcggt ggactacgac ggagactcct acatgaactg gtatcagcag aagcccggaa aagcccca aa gttgctgatc tacgccgcct cataccttga aagcggcgtg cctatgcgct tctcgggaag cgggtcgggc accgatttca ccctgaccat ttcgagactg aggccggagg acttcgcgac ttactactgc caacagtccc acgaggaccc ctatacgttt ggccagggaa ccaaggtcga aatcaag cgt acggtagcgg ccccatctgt cttcatcttc ccgccatctg atgagcagtt gaaatctgga actgcctctg ttgtgtgcct gctgaataac ttctatccca gagaggccaa agtacagtgg aaggtggata acgccccgca atcgggtaac tcccaggaga gtgtcacaga gcaggacagc aagggacagca cctacagcct cagcagcacc ctgacgctga gcaaagcaga ctacgagaaa cacaaagtct acgcctgcga agtcacccat cagggcctga gctcgcccgt cacaaagagc ttcaac aggg gagagtgt

SEQ ID NO:43

FR L3 S64M (PDB) S60M (Kabat)

GVPMRFSGSG SGTDFTLTIS SLQPEDFATY YC

SEQ ID NO:44

FR L3 S81R (PDB) S77r (Kabat)

GVPSRFSGSG SGTDFTLTIS RLQPEDFATY YC

SEQ ID NO:45

FR L3 Q83R (PDB) q79r (Kabat)

GVPSRFSGSG SGTDFTLTIS SLRPEDFATY YC

SEQ ID NO:46

FR L3 S64M S81R (PDB) S60M S77R (Kabat)

GVPMRFSGSG SGTDFTLTIS RLQPEDFATY YC

SEQ ID NO:47

FR L3 S64M Q83R (PDB) S60M Q79R (Kabat)

GVPMRFSGSG SGTDFTLTIS SLRPEDFATY YC

SEQ ID NO:48

FR L3 S81R Q83R (PDB) S77R Q79R (Kabat)

GVPSRFSGSG SGTDFTLTIS RLRPEDFATY YC

SEQ ID NO:49

FR L3 S64M S81R Q83R (PDB) S60M S77R Q79R (Kabat)

GVPMRFSGSG SGTDFTLTIS RLRPEDFATY YC

SEQ ID NO:50

CDRL2 S56D (PDB) S52d (Kabat)

aaDyles

SEQ ID NO:51

CDRL2 S56E (PDB) S52e (Kabat)

aaEyles

SEQ ID NO:52

FR L3 S71M (PDB) S67m (Kabat)

GVPSRFSGSG SGTDFTLTIS SLQPEDFATY YC

SEQ ID NO:53

FR L3 S64M S71M (PDB) S60M S67m (Kabat)

GVPMRFSGSG MGTDFTLTIS SLQPEDFATY YC

SEQ ID NO:54

FR L3 S81R S71M (PDB) S77r S67m (Kabat)

GVPSRFSGSG MGTDFTLTIS RLQPEDFATY YC

SEQ ID NO:55

FR L3 Q83R S71M (PDB) q79r S67m (Kabat)

GVPSRFSGSG MGTDFTLTIS SLRPEDFATY YC

SEQ ID NO:56

FR L3 S64M S81R S71M (PDB) S60M S77R S67m (Kabat)

GVPMRFSGSG MGTDFTLTIS RLQPEDFATY YC

SEQ ID NO:57

FR L3 S64M Q83R S71M (PDB) S60M Q79R S67m (Kabat)

GVPMRFSGSG MGTDFTLTIS SLRPEDFATY YC

SEQ ID NO:58

FR L3 S81R Q83R S71M (PDB) S77R Q79R S67m (Kabat)

GVPSRFSGSG MGTDFTLTIS RLRPEDFATY YC

SEQ ID NO:59

FR L3 S64M S81R Q83R S71M (PDB) S60M S77R Q79R S67m (Kabat)

GVPMRFSGSG MGTDFTLTIS RLRPEDFATY YC

SEQ ID NO:60

FR L3 S67Y (PDB) S63Y (Kabat)

GVPSRFYGSG SGTDFTLTIS SLQPEDFATY YC

SEQ ID NO:61

FR L3 S64M S67Y (PDB) S60M S63Y (Kabat)

GVPMRFYGSG SGTDFTLTIS SLQPEDFATY YC

SEQ ID NO:62

FR L3 S81R S67Y (PDB) S77r S63Y (Kabat)

GVPSRFYGSG SGTDFTLTIS RLQPEDFATY YC

SEQ ID NO:63

FR L3 Q83R S67Y (PDB) q79r S63Y (Kabat)

GVPSRFYGSG SGTDFTLTIS SLRPEDFATY YC

SEQ ID NO:64

FR L3 S64M S81R S67Y (PDB) S60M S77R S63Y (Kabat)

GVPMRFYGSG SGTDFTLTIS RLQPEDFATY YC

SEQ ID NO:65

FR L3 S64M Q83R S67Y (PDB) S60M Q79R S63Y (Kabat)

GVPMRFYGSG SGTDFTLTIS SLRPEDFATY YC

SEQ ID NO:66

FR L3 S81R Q83R S67Y (PDB) S77R Q79R S63Y (Kabat)

GVPSRFYGSG SGTDFTLTIS RLRPEDFATY YC

SEQ ID NO:67

FR L3 S64M S81R Q83R S67Y (PDB) S60M S77R Q79R S63Y (Kabat)

GVPMRFYGSG SGTDFTLTIS RLRPEDFATY YC

SEQ ID NO:68

FR L3 S80N (PDB) S76N (Kabat)

GVPSRFSGSG SGTDFTLTIN SLQPEDFATY YC

SEQ ID NO:69

FR L3 S64M S80N (PDB) S60M S76N (Kabat)

GVPMRFSGSG SGTDFTLTIN SLQPEDFATY YC

SEQ ID NO:70

FR L3 S81R S80N (PDB) S77r S76N (Kabat)

GVPSRFSGSG SGTDFTLTIN RLQPEDFATY YC

SEQ ID NO:71

FR L3 Q83R S80N (PDB) q79r S76N (Kabat)

GVPSRFSGSG SGTDFTLTIN SLRPEDFATY YC

SEQ ID NO:72

FR L3 S64M S81R S80N (PDB) S60M S77R S76N (Kabat)

GVPMRFSGSG SGTDFTLTIN RLQPEDFATY YC

SEQ ID NO:73

FR L3 S64M Q83R S80N (PDB) S60M Q79R S76N (Kabat)

GVPMRFSGSG SGTDFTLTIN SLRPEDFATY YC

SEQ ID NO:74

FR L3 S81R Q83R S80N (PDB) S77R Q79R S76N (Kabat)

GVPSRFSGSG SGTDFTLTIN RLRPEDFATY YC

SEQ ID NO:75

FR L3 S64M S81R Q83R S80N (PDB) S60M S77R Q79R S76N (Kabat)

GVPMRFSGSG SGTDFTLTIN RLRPEDFATY YC

SEQ ID NO:76

FR L3 S80N S67Y (PDB) S76N S63Y(Kabat)

GVPSRFYGSG SGTDFTLTIN SLQPEDFATY YC

SEQ ID NO:77

FR L3 S64M S80N S67Y (PDB) S60M S76N S63Y (Kabat)

GVPMRFYGSG SGTDFTLTIN SLQPEDFATY YC

SEQ ID NO:78

FR L3 S81R S80N S67Y (PDB) S77r S76N S63Y (Kabat)

GVPSRFYGSG SGTDFTLTIN RLQPEDFATY YC

SEQ ID NO:79

FR L3 Q83R S80N S67Y (PDB) q79r S76N S63Y (Kabat)

GVPSRFYGSG SGTDFTLTIN SLRPEDFATY YC

SEQ ID NO:80

FR L3 S64M S81R S80N S67Y (PDB) S60M S77R S76N S63Y (Kabat)

GVPMRFYGSG SGTDFTLTIN RLQPEDFATY YC

SEQ ID NO:81

FR L3 S64M Q83R S80N S67Y (PDB) S60M Q79R S76N S63Y (Kabat)

GVPMRFYGSG SGTDFTLTIN SLRPEDFATY YC

SEQ ID NO:82

FR L3 S81R Q83R S80N S67Y (PDB) S77R Q79R S76N S63Y (Kabat)

GVPSRFYGSG SGTDFTLTIN RLRPEDFATY YC

SEQ ID NO:83

FR L3 S64M S81R Q83R S80N S67Y (PDB) S60M S77R Q79R S76N S63Y (Kabat)

GVPMRFYGSG SGTDFTLTIN RLRPEDFATY YC

SEQ ID NO:84

FR L3 S67Y S71M (PDB) S63Y S67M (Kabat)

GVPSRFYGSG MGTDFTLTIS SLQPEDFATY YC

SEQ ID NO:85

FR L3 S64M S67Y S71M (PDB) S60M S63Y S67M (Kabat)

GVPMRFYGSG MGTDFTLTIS SLQPEDFATY YC

SEQ ID NO:86

FR L3 S81R S67Y S71M (PDB) S77r S63Y S67M (Kabat)

GVPSRFYGSG MGTDFTLTIS RLQPEDFATY YC

SEQ ID NO:87

FR L3 Q83R S67Y S71M (PDB) q79r S63Y S67M (Kabat)

GVPSRFYGSG MGTDFTLTIS SLRPEDFATY YC

SEQ ID NO:88

FR L3 S64M S81R S67Y S71M (PDB) S60M S77R S63Y S67M (Kabat)

GVPMRFYGSG MGTDFTLTIS RLQPEDFATY YC

SEQ ID NO:89

FR L3 S64M Q83R S67Y S71M (PDB) S60M Q79R S63Y S67M (Kabat)

GVPMRFYGSG MGTDFTLTIS SLRPEDFATY YC

SEQ ID NO:90

FR L3 S81R Q83R S67Y S71M (PDB) S77R Q79R S63Y S67M (Kabat)

GVPSRFYGSG MGTDFTLTIS RLRPEDFATY YC

SEQ ID NO:91

FR L3 S64M S81R Q83R S67Y S71M (PDB) S60M S77R Q79R S63Y S67M (Kabat)

GVPMRFYGSG MGTDFTLTIS RLRPEDFATY YC

SEQ ID NO:92

FR L3 S80N S71M (PDB) S76N S67M (Kabat)

GVPSRFSGSG MGTDFTLTIN SLQPEDFATY YC

SEQ ID NO:93

FR L3 S64M S80N S71M (PDB) S60M S76N S67M (Kabat)

GVPMRFSGSG MGTDFTLTIN SLQPEDFATY YC

SEQ ID NO:94

FR L3 S81R S80N S71M (PDB) S77r S76N S67M (Kabat)

GVPSRFSGSG MGTDFTLTIN RLQPEDFATY YC

SEQ ID NO:95

FR L3 Q83R S80N S71M (PDB) q79r S76N S67M (Kabat)

GVPSRFSGSG MGTDFTLTIN SLRPEDFATY YC

SEQ ID NO:96

FR L3 S64M S81R S80N S71M (PDB) S60M S77R S76N S67M (Kabat)

GVPMRFSGSG MGTDFTLTIN RLQPEDFATY YC

SEQ ID NO:97

FR L3 S64M Q83R S80N S71M (PDB) S60M Q79R S76N S67M (Kabat)

GVPMRFSGSG MGTDFTLTIN SLRPEDFATY YC

SEQ ID NO:98

FR L3 S81R Q83R S80N S71M (PDB) S77R Q79R S76N S67M (Kabat)

GVPSRFSGSG MGTDFTLTIN RLRPEDFATY YC

SEQ ID NO:99

FR L3 S64M S81R Q83R S80N S71M (PDB) S60M S77R Q79R S76N S67M (Kabat)

GVPMRFSGSG MGTDFTLTIN RLRPEDFATY YC

SEQ ID NO:100

FR L3 S80N S67Y S71M (PDB) S76N S63Y S67M (Kabat)

GVPSRFYGSG MGTDFTLTIN SLQPEDFATY YC

SEQ ID NO:101

FR L3 S64M S80N S67Y S71M (PDB) S60M S76N S63Y S67M (Kabat)

GVPMRFYGSG MGTDFTLTIN SLQPEDFATY YC

SEQ ID NO:102

FR L3 S81R S80N S67Y S71M (PDB) S77r S76N S63Y S67M (Kabat)

GVPSRFYGSG MGTDFTLTIN RLQPEDFATY YC

SEQ ID NO:103

FR L3 Q83R S80N S67Y S71M (PDB) q79r S76N S63Y S67M (Kabat)

GVPSRFYGSG MGTDFTLTIN SLRPEDFATY YC

SEQ ID NO:104

FR L3 S64M S81R S80N S67Y S71M (PDB) S60M S77R S76N S63Y S67M (Kabat)

GVPMRFYGSG MGTDFTLTIN RLQPEDFATY YC

SEQ ID NO:105

FR L3 S64M Q83R S80N S67Y S71M (PDB) S60M Q79R S76N S63Y S67M (Kabat)

GVPMRFYGSG MGTDFTLTIN SLRPEDFATY YC

SEQ ID NO:106

FR L3 S81R Q83R S80N S67Y S71M (PDB) S77R Q79R S76N S63Y S67M (Kabat)

GVPSRFYGSG MGTDFTLTIN RLRPEDFATY YC

SEQ ID NO:107

FR L3 S64M S81R Q83R S80N S67Y S71M (PDB) S60M S77R Q79R S76N S63Y S67M (Kabat)

GVPMRFYGSG MGTDFTLTIN RLRPEDFATY YC

SEQ ID NO:108

Wild-type human IgE-Fc (domains Cε2-Cε4 numbered V224-K547 according to Dorrington & Bennich (1978) Immunol. Rev. 41:3-25), carrying the C225A mutation; Note, in crystallographic experiments the following mutations were also introduced into IgE-Fc to simplify the glycosylation pattern: N265Q and N371Q (also following Dorrington & Bennich numbering)

VASRDFTPPT VKILQSSCDG GGHFPPTIQL LCLVSGYTPG TINITWLEDG QVMDVDLSTA STTQEGELAS TQSELTLSQK HWLSDRTYTC QVTYQGHTFE DSTKKCADSN PRGVSAYLSR PSPFDLFIRK SPTITCLVVD LAPSKGTVNL TWSRASGKPV NHSTRKEEKQ RNGTLTVTST LPVGTRDWIE GETY QCRVTH PHLPRALMRS TTKTSGPRAA PEVYAFATPE WPGSRDKRTL ACLIQNFMPE DISVQWLHNE VQLPDARHST TQPRKTKGSG FFVFSRLEVT RAEWEQKDEF ICRAVHEAAS PSQTVQRAVS VNPGK

LIGHT CHAIN

Fab1

SEQ ID NO:109

S77R_Q79R_v-area (Fab1)

DIQLTQSPSS LSASVGDRVT ITCRASQSVD YDGDSYMNWY QQKPGKAPKL LIYAASYLES GVPSRFSGSG SGTDFTLTIS RLRPEDFATY YCQQSHEDPY TFGQGTKVEI K

SEQ ID NO:110

S77R_Q79R_v-area (Fab1)

GATATTCAGC TGACTCAGAG CCCGAGCTCA CTCTCCGCTT CCGTGGGGGA TAGAGTGACC ATCACTTGCC GGGCATCCCA GTCGGTGGAC TACGACGGAG ACTCCTACAT GAACTGGTAC CAGCAGAAGC CCGGAAAAGC CCCAAAGTTG CTGATCTACG CCGCCTCCTA CCTTGAAAGC GGCGTGCCTT CACGCTTCTC GGGAAGCGGG TCTGGCACCG ATTT CACCCT GACCATTTCG AGACTGAGGC CGGAGGACTT CGCGACTTAC TACTGCCAAC AGTCCCACGA GGACCCCTAT ACGTTTGGCC AGGGTACCAA GGTCGAAATC AAG

SEQ ID NO:111

S77R_Q79R_v-region (Fab1) with signal sequence, which is underlined and italicized

mkwvtfisll flfssaysdi qltqspssls asvgdrvtit crasqsvdyd gdsymnwyqq kpgkapklli yaasylesgv pSrfsgsgsg tdftltisrl rpedfatyyc qqshedpytf gqgtkveik

SEQ ID NO:112

S77R_Q79R_v-region (Fab1) with signal sequence, which is underlined and italicized

atgaagtggg tcaccttcat ctccctgctg tttctgttct cctccgccta ctccGATATT CAGCTGACTC AGAGCCCGAG CTCACTCTCC GCTTCCGTGG GGGATAGAGT GACCATCACT TGCCGGGCAT CCCAGTCGGT GGACTACGAC GGAGACTCCT ACATGAACTG GTACCAGCAG AAGCCCGGAA AAGCCCCAAA GTTGCTGATC TACG CCGCCT CCTACCTTGA AAGCGGCGTG CCTTCACGCT TCTCGGGAAG CGGGTCTGGC ACCGATTTCA CCCTGACCAT TTCGAGACTG AGGCCGGAGG ACTTCGCGAC TTACTACTGC CAACAGTCCC ACGAGGACCC CTATACGTTT GGCCAGGGTA CCAAGGTCGA AATCAAG

Fab2

Omalizumab_v-region+kappa constant region including L154P

SEQ ID NO:113

Fab2_v-region

DIQLTQSPSS LSASVGDRVT ITCRASQSVD YDGDSYMNWY QQKPGKAPKL LIYAASYLES GVPSRFSGSG SGTDFTLTIS SLQPEDFATY YCQQSHEDPY TFGQGTKVEI K

SEQ ID NO:114

Fab2_v-region

gatattcagc tgactcagag cccgagctca ctctccgctt ccgtggggga tagagtgacc atcacttgcc gggcatccca gtcggtggac tacgacggag actcctacat gaactggtat cagcagaagc ccggaaaagc cccaaagttg ctgatctacg ccgcctcata ccttgaaagc ggcgtgcctt cg cgcttctc gggaagcggg tcgggcaccg atttcaccct gaccatttcg tcgctgcagc cggaggactt cgcgacttac tactgccaac agtcccacga ggacccctat acgtttggcc agggaaccaa ggtcgaaatc aag

SEQ ID NO:115

Fab2_v region with signal sequence, which is underlined and italicized

mkwvtfisll flfssaysdi qltqspssls asvgdrvtit crasqsvdyd gdsymnwyqq kpgkapklli yaasylesgv pSrfsgsgsg tdftltisSl Qpedfatyyc qqshedpytf gqgtkveik

SEQ ID NO:116

Fab2_v region with signal sequence, which is underlined and italicized

atgaagtggg tcaccttcat ctccctgctg tttctgttct cctccgccta ctccgatatt cagctgactc agagcccgag ctcactctcc gcttccgtgg gggatagagt gaccatcact tgccgggcat cccagtcggt ggactacgac ggagactcct acatgaactg gtatcagcag aagcccggaa aagcccca aa gttgctgatc tacgccgcct cataccttga aagcggcgtg ccttcgcgct tctcgggaag cgggtcgggc accgatttca ccctgaccat ttcgtcgctg cagccggagg acttcgcgac ttactactgc caacagtccc acgaggaccc ctatacgttt ggccagggaa ccaaggtcga a atcaag

SEQ ID NO:117

Fab2_v region + kappa constant region including L154P

DIQLTQSPSS LSASVGDRVT ITCRASQSVD YDGDSYMNWY QQKPGKAPKL LIYAASYLES GVPSRFSGSG SGTDFTLTIS SLQPEDFATY YCQQSHEDPY TFGQGTKVEI KRTVAAPSVF IFPPSDEQLK SGTASVVCLL NNFYPREAKV QWKVDNAPQS GNSQESVTEQ DSKDSTYSLS STLTLSKADY EKHKVYACEV THQGLSSPVT KSFNRGEC

SEQ ID NO:118

Fab2_v region + kappa constant region including L154P

gatattcagc tgactcagag cccgagctca ctctccgctt ccgtggggga tagagtgacc atcacttgcc gggcatccca gtcggtggac tacgacggag actcctacat gaactggtat cagcagaagc ccggaaaagc cccaaagttg ctgatctacg ccgcctcata ccttgaaagc ggcgtgcctt cg cgcttctc gggaagcggg tcgggcaccg atttcaccct gaccatttcg tcgctgcagc cggaggactt cgcgacttac tactgccaac agtcccacga ggacccctat acgtttggcc agggaaccaa ggtcgaaatc aagcgtacgg tagcggcccc atctgtcttc atcttcccgc catctgatga gca gttgaaa tctggaactg cctctgttgt gtgcctgctg aataacttct atcccagaga ggccaaagta cagtggaagg tggataacgc cccgcaatcg ggtaactccc aggagagtgt cacagagcag gacagcaagg acagcaccta cagcctcagc agcaccctga cgctgagcaa agcagactac gagaaacaca aagtctacgc ctgcgaagtc acccatcagg gcctgagctc gcccgtcaca aagagcttca acaggggaga gtgt

SEQ ID NO:119

Fab2_v region + kappa constant region including L154P, with signal sequence underlined and italicized

mkwvtfisll flfssaysDI QLTQSPSSLS ASVGDRVTIT CRASQSVDYD GDSYMNWYQQ KPGKAPKLLI YAASYLESGV PSRFSGSGSG TDFTLTISSL QPEDFATYYC QQSHEDPYTF GQGTKVEIKR TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNAPQSGN SQ ESVTEQDS KDSTYSLSST LTLSKADYEK HKVYACEVTH QGLSSPVTKS FNRGEC

SEQ ID NO:120

Fab2_v region + kappa constant region including L154P, with signal sequence underlined and italicized

atgaagtggg tcaccttcat ctccctgctg tttctgttct cctccgccta ctccgatatt cagctgactc agagcccgag ctcactctcc gcttccgtgg gggatagagt gaccatcact tgccgggcat cccagtcggt ggactacgac ggagactcct acatgaactg gtatcagcag aagcccggaa aagcccca aa gttgctgatc tacgccgcct cataccttga aagcggcgtg ccttcgcgct tctcgggaag cgggtcgggc accgatttca ccctgaccat ttcgtcgctg cagccggagg acttcgcgac ttactactgc caacagtccc acgaggaccc ctatacgttt ggccagggaa ccaaggtcga a atcaagcgt acggtagcgg ccccatctgt cttcatcttc ccgccatctg atgagcagtt gaaatctgga actgcctctg ttgtgtgcct gctgaataac ttctatccca gagaggccaa agtacagtgg aaggtggata acgccccgca atcgggtaac tcccaggaga gtgtcacaga gcaggacagc aagggacagca cctacagcct cagcagcacc ctgacgctga gcaaagcaga ctacgagaaa cacaaagtct acgcctgcga agtcacccat cagggcctga gctcgcccgt cacaaagagc ttcaac aggg gagagtgt

Fab3

S77R_Q79R_v-region (from Fab1) + kappa constant region including L154P (from Fab2)

SEQ ID NO:121

S77R_Q79R_v-area

DIQLTQSPSS LSASVGDRVT ITCRASQSVD YDGDSYMNWY QQKPGKAPKL LIYAASYLES GVPSRFSGSG SGTDFTLTIS RLRPEDFATY YCQQSHEDPY TFGQGTKVEI K

SEQ ID NO:122

S77R_Q79R_v-area

GATATTCAGC TGACTCAGAG CCCGAGCTCA CTCTCCGCTT CCGTGGGGGA TAGAGTGACC ATCACTTGCC GGGCATCCCA GTCGGTGGAC TACGACGGAG ACTCCTACAT GAACTGGTAC CAGCAGAAGC CCGGAAAAGC CCCAAAGTTG CTGATCTACG CCGCCTCCTA CCTTGAAAGC GGCGTGCCTT CACGCTTCTC GGGAAGCGGG TCTGGCACCG ATTT CACCCT GACCATTTCG AGACTGAGGC CGGAGGACTT CGCGACTTAC TACTGCCAAC AGTCCCACGA GGACCCCTAT ACGTTTGGCC AGGGTACCAA GGTCGAAATC AAG

SEQ ID NO:123

S77R_Q79R_v - region with signal sequence, which is underlined and italicized

mkwvtfisll flfssaysdi qltqspssls asvgdrvtit crasqsvdyd gdsymnwyqq kpgkapklli yaasylesgv pSrfsgsgsg tdftltisrl rpedfatyyc qqshedpytf gqgtkveik

SEQ ID NO:124

S77R_Q79R_v - region with signal sequence, which is underlined and italicized

atgaagtggg tcaccttcat ctccctgctg tttctgttct cctccgccta ctccGATATT CAGCTGACTC AGAGCCCGAG CTCACTCTCC GCTTCCGTGG GGGATAGAGT GACCATCACT TGCCGGGCAT CCCAGTCGGT GGACTACGAC GGAGACTCCT ACATGAACTG GTACCAGCAG AAGCCCGGAA AAGCCCCAAA GTTGCTGATC TACG CCGCCT CCTACCTTGA AAGCGGCGTG CCTTCACGCT TCTCGGGAAG CGGGTCTGGC ACCGATTTCA CCCTGACCAT TTCGAGACTG AGGCCGGAGG ACTTCGCGAC TTACTACTGC CAACAGTCCC ACGAGGACCC CTATACGTTT GGCCAGGGTA CCAAGGTCGA AATCAAG

SEQ ID NO:125

S77R_Q79R_v-region+kappa constant region including L154P

DIQLTQSPSS LSASVGDRVT ITCRASQSVD YDGDSYMNWY QQKPGKAPKL LIYAASYLES GVPSRFSGSG SGTDFTLTIS RLRPEDFATY YCQQSHEDPY TFGQGTKVEI KRTVAAPSVF IFPPSDEQLK SGTASVVCLL NNFYPREAKV QWKVDNAPQS GNSQESVTEQ DSKDSTYSLS ST LTLSKADY EKHKVYACEV THQGLSSPVT KSFNRGEC

SEQ ID NO:126

S77R_Q79R_v-region+kappa constant region including L154P

GATATTCAGC TGACTCAGAG CCCGAGCTCA CTCTCCGCTT CCGTGGGGGA TAGAGTGACC ATCACTTGCC GGGCATCCCA GTCGGTGGAC TACGACGGAG ACTCCTACAT GAACTGGTAC CAGCAGAAGC CCGGAAAAGC CCCAAAGTTG CTGATCTACG CCGCCTCCTA CCTTGAAAGC GGCGTGCCTT CACGCTTCTC GGGAAGCGGG TCTGGCACCG ATTT CACCCT GACCATTTCG AGACTGAGGC CGGAGGACTT CGCGACTTAC TACTGCCAAC AGTCCCACGA GGACCCCTAT ACGTTTGGCC AGGGTACCAA GGTCGAAATC AAGcgtacgg tagcggcccc atctgtcttc atcttcccgc catctgatga gcagttgaaa tctggaactg cctctgttgt gtgcctgctg aataact tct atcccagaga ggccaaagta cagtggaagg tggataacgc cccgcaatcg ggtaactccc aggagagtgt cacagagcag gacagcaagg acagcaccta cagcctcagc agcaccctga cgctgagcaa agcagactac gagaaacaca aagtctacgc ctgcgaagtc acccatcagg gcctgagctc gcccgtcaca aagagcttca acaggggaga gtgt

SEQ ID NO:127

S77R_Q79R_v-region+kappa constant region including L154P, with signal sequence underlined and italicized

mkwvtfisll flfssaysDI QLTQSPSSLS ASVGDRVTIT CRASQSVDYD GDSYMNWYQQ KPGKAPKLLI YAASYLESGV PSRFSGSGSG TDFTLTISRL RPEDFATYYC QQSHEDPYTF GQGTKVEIKR TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNAPQSGN SQ ESVTEQDS KDSTYSLSST LTLSKADYEK HKVYACEVTH QGLSSPVTKS FNRGEC

SEQ ID NO:128

S77R_Q79R_v-region+kappa constant region including L154P, with signal sequence underlined and italicized

atgaagtggg tcaccttcat ctccctgctg tttctgttct cctccgccta ctccGATATT CAGCTGACTC AGAGCCCGAG CTCACTCTCC GCTTCCGTGG GGGATAGAGT GACCATCACT TGCCGGGCAT CCCAGTCGGT GGACTACGAC GGAGACTCCT ACATGAACTG GTACCAGCAG AAGCCCGGAA AAGCCCCAAA GTTGCTGATC TACG CCGCCT CCTACCTTGA AAGCGGCGTG CCTTCACGCT TCTCGGGAAG CGGGTCTGGC ACCGATTTCA CCCTGACCAT TTCGAGACTG AGGCCGGAGG ACTTCGCGAC TTACTACTGC CAACAGTCCC ACGAGGACCC CTATACGTTT GGCCAGGGTA CCAAGGTCGA AATCAAGcgt acggtagcgg ccccatctgt cttcatcttc ccgccatctg atgag cagtt gaaatctgga actgcctctg ttgtgtgcct gctgaataac ttctatccca gagaggccaa agtacagtgg aaggtggata acgccccgca atcgggtaac tcccaggaga gtgtcacaga gcaggacagc aagggacagca cctacagcct cagcagcacc ctgacgctga gcaaagcaga ctacgagaaa cacaaagtct acgcctgcga agtcacccat cagggcctga gctcgcccgt cacaaagagc ttcaac aggg gagagtgt

SEQ ID NO:129

Omalizumab_v-region

TQSPSSLSAS VGDRVTITCR ASQSVDYDGD SYMNWYQQKP GKAPKLLIYA ASYLESGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQQ SHEDPYTFGQ GTKVEIK

SEQ ID NO:130

Omalizumab_v-region

actcagagcc cgagctcact ctccgcttcc gtgggggata gagtgaccat cacttgccgg gcatcccagt cggtggacta cgacggagac tcctacatga actggtatca gcagaagccc ggaaaagccc caaagttgct gatctacgcc gcctcatacc ttgaaagcgg cgtgccttcg cgcttctcgg gaagcggg tc gggcaccgat ttcaccctga ccatttcgtc gctgcagccg gaggacttcg cgacttacta ctgccaacag tcccacgagg acccctatac gtttggccag ggaaccaagg tcgaaatcaa g

SEQ ID NO:131

FR3

GVPMRFSSGMGGTDFTLTISRLRPEDFATYYC

SEQ ID NO:132

S60M_S52D_S67M_S77R_Q79R_v-area

DIQLTQSPSS LSASVGDRVT ITCRASQSVD YDGDSYMNWY QQKPGKAPKL LIYAADYLES GVPMRFSGSG MGTDFTLTIS RLRPEDFATY YCQQSHEDPY TFGQGTKVEI K

SEQ ID NO:133

S60M_S52D_S67M_S77R_Q79R_v-area

GATATTCAGCTGACTCAGAGCCCGAGCTCACTCTCCGCTTCCGTGGGGGATAGAGTGACCATCACTTGCCGGGCATCCCAGTCGGTGGACTACGACGGAGACTCCTACATGAACTGGTATCAGCAGAAGCCCGGAAAAGCCCCAAAGTTGCTGATCTACGCCGCCGACTACCTTGAAAGCGGCGTGCCTATGCGCTTCTCGGGAAGCGGGATGGGCACCGATTTCACCCTGACCATTTCGAGACTGAGGCCG GAGGACTTCGCGACTTACTACTGCCAACAGTCCCACGAGGACCCCTATACGTTTGGCCAGGGAACCAAGGTCGAAATCAAG

SEQ ID NO:134

S60M_S52D_S67M_S77R_Q79R_v - region with signal sequence, which is underlined and italicized

mkwvtfisll flfssaysdi qltqspssls asvgdrvtit crasqsvdyd gdsymnwyqq kpgkapklli yaaDylesgv pMrfsgsgMg tdftltisrl rpedfatyyc qqshedpytf gqgtkveik

SEQ ID NO:135

S60M_S52D_S67M_S77R_Q79R_v - region with signal sequence, which is underlined and italicized

ATGAAGTGGGTCACCTTCATCTCCCTGCTGTTTCTGTTCTCCTCCGCCTACTCCGATATTCAGCTGACTCAGAGCCCGAGCTCACTCTCCGCTTCCGTGGGGGATAGAGTGACCATCACTTGCCGGGCATCCCAGTCGGTGGACTACGACGGAGACTCCTACATGAACTGGTATCAGCAGAAGCCCGGAAAAGCCCCAAAGTTGCTGATCTACGCCGCCGACTACCTTGAAAGCGGCGTGCCTATGCGCTTC TCGGGAAGCGGGATGGGCACCGATTTCACCCTGACCATTTCGAGACTGAGGCCGGAGGACTTCGCGACTTACTACTGCCAACAGTCCCACGAGGACCCCTATACGTTTGGCCAGGGAACCAAGGTCGAAATCAAG

SEQ ID NO:136

S60M_S52D_S67M_S77R_Q79R_v-region+kappa constant region

DI QLTQSPSSLS ASVGDRVTIT CRASQSVDYD GDSYMNWYQQ KPGKAPKLLI YAADYLESGV PMRFSGSGMG TDFTLTISRL RPEDFATYYC QQSHEDPYTF GQGTKVEIKR TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN SQESVTEQDS KDSTYSLSST LTLSKADYEK HKVYACEVTH QGLSSPVTKS FNRGEC

SEQ ID NO:137

S60M_S52D_S67M_S77R_Q79R_v-region+kappa constant region

GATATTCAGCTGACTCAGAGCCCGAGCTCACTCTCCGCTTCCGTGGGGGATAGAGTGACCATCACTTGCCGGGCATCCCAGTCGGTGGACTACGACGGAGACTCCTACATGAACTGGTATCAGCAGAAGCCCGGAAAAGCCCCAAAGTTGCTGATCTACGCCGCCGACTACCTTGAAAGCGGCGTGCCTATGCGCTTCTCGGGAAGCGGGATGGGCACCGATTTCACCCTGACCATTTCGAGACTGAGGCCG GAGGACTTCGCGACTTACTACTGCCAACAGTCCCACGAGGACCCCTATACGTTTGGCCAGGGAACCAAGGTCGAAATCAAGCGTACGGTAGCGGCCCCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTC ACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT

SEQ ID NO:138

FR3

GVPRRFSGSMGMGTDFTLTISRLRPEDFATYYC

SEQ ID NO:139

S60R_S52D_S67M_S77R_Q79R_v-area

DIQLTQSPSS LSASVGDRVT ITCRASQSVD YDGDSYMNWY QQKPGKAPKL LIYAADYLES GVPRRFSGSG MGTDFTLTIS RLRPEDFATY YCQQSHEDPY TFGQGTKVEI K

SEQ ID NO:140

S60R_S52D_S67M_S77R_Q79R_v-area

gatattcagctgactcagagcccgagctcactctccgcttccgtgggggatagagtgaccatcacttgccgggcatcccagtcggtggactacgacggagactcctacatgaactggtatcagcagaagcccggaaaagccccaaagttgctgatctacgccgccgattaccttgaaagcggcgtgcctcgtcgcttctcgggaagcgg gatgggcaccgatttcaccctgaccatttcgagactgaggccggaggacttcgcgacttactactgccaacagtcccacgaggacccctatacgtttggccagggaaccaaggtcgaaatcaag

SEQ ID NO:141

S60R_S52D_S67M_S77R_Q79R_v - region with signal sequence, which is underlined and italicized

mkwvtfisll flfssaysdi qltqspssls asvgdrvtit crasqsvdyd gdsymnwyqq kpgkapklli yaaDylesgv pRrfsgsgMg tdftltisrl rpedfatyyc qqshedpytf gqgtkveik

SEQ ID NO:142

S60R_S52D_S67M_S77R_Q79R_v - region with signal sequence, which is underlined and italicized

ATGAAGTGGGTCACCTTCATCTCCCTGCTGTTTCTGTTCTCCTCCGCCTACTCCGATATTCAGCTGACTCAGAGCCCGAGCTCACTCTCCGCTTCCGTGGGGGATAGAGTGACCATCACTTGCCGGGCATCCCAGTCGGTGGACTACGACGGAGACTCCTACATGAACTGGTATCAGCAGAAGCCCGGAAAAGCCCCAAAGTTGCTGATCTACGCCGCCGATTACCTTGAAAGCGGCGTGCCTCGTCGCTT CTCGGGAAGCGGGATGGGCACCGATTTCACCCTGACCATTTCGAGACTGAGGCCGGAGGACTTCGCGACTTACTACTGCCAACAGTCCCACGAGGACCCCTATACGTTTGGCCAGGGAACCAAGGTCGAAATCAAG

SEQ ID NO:143

S60R_S52D_S67M_S77R_Q79R_v-region+kappa constant region

DI QLTQSPSSLS ASVGDRVTIT CRASQSVDYD GDSYMNWYQQ KPGKAPKLLI YAADYLESGV PRRFSGSGMG TDFTLTISRL RPEDFATYYC QQSHEDPYTF GQGTKVEIKR TVAAPSVFIF PPSDEQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN SQESVTEQDS KDSTYSLSST LTLSKADYEK HKVYACEVTH QGLSSPVTKS FNRGEC

SEQ ID NO:144

S60R_S52D_S67M_S77R_Q79R_v-region+kappa constant region

gatattcagctgactcagagcccgagctcactctccgcttccgtgggggatagagtgaccatcacttgccgggcatcccagtcggtggactacgacggagactcctacatgaactggtatcagcagaagcccggaaaagccccaaagttgctgatctacgccgccgattaccttgaaagcggcgtgcctcgtcgcttctcgggaagcgg gatgggcaccgatttcaccctgaccatttcgagactgaggccggaggacttcgcgacttactactgccaacagtcccacgaggacccctatacgtttggccagggaaccaaggtcgaaatcaagcgtacggtagcggccccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctg ctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaagggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaaga gcttcaacaggggagagtgt

SEQ ID NO:145

S60K_S52D_S67M_S77R_Q79R_v-area (Kabat)

DIQLTQSPSS LSASVGDRVT ITCRASQSVD YDGDSYMNWY QQKPGKAPKL LIYAADYLES GVPKRFSGSG MGTDFTLTIS RLRPEDFATY YCQQSHEDPY TFGQGTKVEI K

SEQ ID NO:146

S60Q_S52D_S67M_S77R_Q79R_v-area (Kabat)

DIQLTQSPSS LSASVGDRVT ITCRASQSVD YDGDSYMNWY QQKPGKAPKL LIYAADYLES GVPQRFSGSG MGTDFTLTIS RLRPEDFATY YCQQSHEDPY TFGQGTKVEI K

SEQ ID NO:147

v-region+CH1 gamma-1 constant region plus linker plus CA645 gL4gH5 scFv with signal sequence underlined and italicized

MKWVTFISLLFLFSSAYSEVQLVESGGGLVQPGGSLRLSCAVSGYSITSGYSWNWIRQAPGKGLEWVASITYDGSTNYNPSVKGRITISRDDSKNTFYLQMNSLRAEDTAVYYCARGSHYFGHWHFAVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCSGGGGTGGGGSEVQLLESGGGLVQPGGSLRLSCAVSGIDLSNYAINWVRQAPGKCLEWIGIIWASGTTFYATWAKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCARTVPGYSTAPYFDLWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSVSAG DRVTITCQSSPSVWSNFLSWYQQKPGKAPKLLIYEASKLTSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCGGGYSSISDTTFGCGTKVEIKRT

SEQ ID NO:148

v-region+CH1 gamma-1 constant region plus linker plus CA645 gL4gH5 scFv with signal sequence underlined and italicized

atgaagtgggtcaccttcatctccctgctgtttctgttctccagcgcctactccgaagtgcagttggtggagtcgggtggagggctggtgcagcctggcggtagcctgaggctgtcctgtgccgtgtccggatactccattacctccggctactcgtggaactggatcagacaggctcccggaaagggacttgagtggggtgg cgtccatcacctacgacggctcaaccaactataacccgtccgtgaagggccgcatcaccatttcgcgcgacgacagcaagaatactttttacctccaaatgaacagcctgcgggccgaagatactgccgtgtactactgcgcgcggggatcacattacttcgggcactggcacttcgccgtctggggacagggcaccctcgtcactgtct cgagcgcttctacaaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccagtgacggtgtcgtggaactcaggtgccctgaccagcggcgttcacaccttcccggctgtcctacagtcttcaggactctactccctgagcagcgt ggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtcgataagaaagttgagcccaaatcttgtagtggaggtgggggcaccggtggaggtggcagcgaggttcaactgcttgagtctggaggaggcctagtccagcctggagggagcctgcgtctctcttgtgca gtaagcggcatcgacctgagcaattacgccatcaactgggtgagacaagctccggggaagtgtttagaatggatcggtataatatgggccagtgggacgaccttttatgctacatgggcgaaaggaaggtttacaattagccgggacaatagcaaaaacaccgtgtatctccaaatgaactccttgcgagcagaggacacggcggtgtactatt gtgctcgcactgtcccaggttatagcactgcaccctacttcgatctgtggggacaagggaccctggtgactgtttcaagtggcggagggggtagtggagggggtggctctgggggtggcggaagcggtggcgggggttctgacatacaaatgactcagtctccttcatcggtatccgcgtccgttggcgatagggtgactattacat gtcaaagctctcctagcgtctggagcaattttctatcctggtatcaacagaaaccggggaaggctccaaaacttctgatttatgaagcctcgaaactcaccagtggagttccgtcaagattcagtggctctggatcagggacagacttcacgttgacaatcagttcgctgcaaccagaggactttgcgacctactattgtggtggaggttacag tagcataagtgatacgacatttgggtgcggtactaaggtggaaatcaaacgtacc

SEQ ID NO:149

Linker between CH1 and CA645 gL4gH5 scFv

SGGGGTGGGGS

SEQ ID NO:150

CA645 gL4gH5 scFv

EVQLLESGGGLVQPGGSLRLSCAVSGIDLSNYAINWVRQAPGKCLEWIGIIWASGTTFYATWAKGRFTISRDNSKNTVYLQMNSLRAEDTAVYYCARTVPGYSTAPYFDLWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSVSASVGDRVTITCQSSPSVWSNFLSWYQQKPGKAPKLLIYEASKLTSGVPSR FSGSGSGTDFTLTISSLQPEDFATYYCGGGYSSISDTTFGCGTKVEIKRT

SEQ ID NO:151

Linker between CA645 gH5 and CA645 gL4 in scFv

GGGGSGGGGSGGGGSGGGGS

SEQ ID NO:152

CA645 CDRH1

GIDLSNYAIN

SEQ ID NO:153

CA645 CDRH2

IIWASGTTFYATWAKG

SEQ ID NO:154

CA645 CDRH3

TVPGYSTAPYFDL

SEQ ID NO:155

CA645CDRL1

QSSPSVWSNFLS

SEQ ID NO:156

CA645CDRL2

EASKLTS

SEQ ID NO:157

CA645CDRL3

GGGYSSISDTT

SEQ ID NO:158

S60M_S52D_S67M_S77R_Q79R_S63W_S76N_v-area (Kabat)

DIQLTQSPSS LSASVGDRVT ITCRASQSVD YDGDSYMNWY QQKPGKAPKL LIYAADYLES GVPMRFWGSG MGTDFTLTIN RLRPEDFATY YCQQSHEDPY TFGQGTKVEI K

SEQ ID NO:159

S60M_S52D_S67M_S77R_Q79R_S63Y_S76N_v-area (Kabat)

DIQLTQSPSS LSASVGDRVT ITCRASQSVD YDGDSYMNWY QQKPGKAPKL LIYAADYLES GVPMRFYGSG MGTDFTLTIN RLRPEDFATY YCQQSHEDPY TFGQGTKVEI K

SEQ ID NO:160

Signal sequence

mkwvtfisll flfssays

--->

LIST OF SEQUENCES

<110> USB Biopharma SPRL

<120> ANTI-IgE ANTIBODIES

<130>PF0087-WO

<150> GB1610198.2

<151> 2016-06-10

<160> 160

<170> Patent In version 3.5

<210> 1

<211> 121

<212> PROTEIN

<213> Artificial sequence

<220>

<223> V-region

<400> 1

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Tyr Ser Ile Thr Ser Gly

20 25 30

Tyr Ser Trp Asn Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp

35 40 45

Val Ala Ser Ile Thr Tyr Asp Gly Ser Thr Asn Tyr Asn Pro Ser Val

50 55 60

Lys Gly Arg Ile Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Phe Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Ser His Tyr Phe Gly His Trp His Phe Ala Val Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 2

<211> 363

<212> DNA

<213> Artificial sequence

<220>

<223> V-region

<400> 2

gaagtgcagt tggtggagtc gggtggaggg ctggtgcagc ctggcggtag cctgaggctg 60

tcctgtgccg tgtccggata ctccattacc tccggctact cgtggaactg gatcagacag 120

gctcccggaa agggacttga gtgggtggcg tccatcacct acgacggctc aaccaactat 180

aacccgtccg tgaagggccg catcaccatt tcgcgcgacg acagcaagaa tactttttac 240

ctccaaatga acagcctgcg ggccgaagat actgccgtgt actactgcgc gcggggatca 300

cattacttcg ggcactggca cttcgccgtc tggggacagg gcaccctcgt cactgtctcg 360

agc 363

<210> 3

<211> 139

<212> PROTEIN

<213> Artificial sequence

<220>

<223> V-region with signal sequence

<400> 3

Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala

1 5 10 15

Tyr Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro

20 25 30

Gly Gly Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Tyr Ser Ile Thr

35 40 45

Ser Gly Tyr Ser Trp Asn Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu

50 55 60

Glu Trp Val Ala Ser Ile Thr Tyr Asp Gly Ser Thr Asn Tyr Asn Pro

65 70 75 80

Ser Val Lys Gly Arg Ile Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

85 90 95

Phe Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

100 105 110

Tyr Cys Ala Arg Gly Ser His Tyr Phe Gly His Trp His Phe Ala Val

115 120 125

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

130 135

<210> 4

<211> 417

<212> DNA

<213> Artificial sequence

<220>

<223> V-region with signal sequence

<400> 4

atgaagtggg tcaccttcat ctccctgctg tttctgttct ccagcgccta ctccgaagtg 60

cagttggtgg agtcgggtgg agggctggtg cagcctggcg gtagcctgag gctgtcctgt 120

gccgtgtccg gatactccat tacctccggc tactcgtgga actggatcag acaggctccc 180

ggaaagggac ttgagtgggt ggcgtccatc acctacgacg gctcaaccaa ctataacccg 240

tccgtgaagg gccgcatcac catttcgcgc gacgacagca agaatacttt ttacctccaa 300

atgaacagcc tgcgggccga agatactgcc gtgtactact gcgcgcgggg atcacattac 360

ttcgggcact ggcacttcgc cgtctgggga cagggcaccc tcgtcactgt ctcgagc 417

<210> 5

<211> 224

<212> PROTEIN

<213> Artificial sequence

<220>

<223> v-region + CH1 gamma-1 constant region

<400> 5

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Tyr Ser Ile Thr Ser Gly

20 25 30

Tyr Ser Trp Asn Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp

35 40 45

Val Ala Ser Ile Thr Tyr Asp Gly Ser Thr Asn Tyr Asn Pro Ser Val

50 55 60

Lys Gly Arg Ile Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Phe Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Ser His Tyr Phe Gly His Trp His Phe Ala Val Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

<210> 6

<211> 672

<212> DNA

<213> Artificial sequence

<220>

<223> v-region + CH1 gamma-1 constant region

<400> 6

gaagtgcagt tggtggagtc gggtggaggg ctggtgcagc ctggcggtag cctgaggctg 60

tcctgtgccg tgtccggata ctccattacc tccggctact cgtggaactg gatcagacag 120

gctcccggaa agggacttga gtgggtggcg tccatcacct acgacggctc aaccaactat 180

aacccgtccg tgaagggccg catcaccatt tcgcgcgacg acagcaagaa tactttttac 240

ctccaaatga acagcctgcg ggccgaagat actgccgtgt actactgcgc gcggggatca 300

cattacttcg ggcactggca cttcgccgtc tggggacagg gcaccctcgt cactgtctcg 360

agcgcttcta caaagggccc atcggtcttc cccctggcac cctcctccaa gagcacctct 420

gggggcacag cggccctggg ctgcctggtc aaggactact tccccgaacc ggtgacggtg 480

tcgtggaact caggcgccct gaccagcggc gtgcacacct tcccggctgt cctacagtcc 540

tcaggactct actccctcag cagcgtggtg accgtgccct ccagcagctt gggcacccag 600

acctacatct gcaacgtgaa tcacaagccc agcaacacca aggtggacaa gaaagttgag 660

cccaaatctt gt 672

<210> 7

<211> 242

<212> PROTEIN

<213> Artificial sequence

<220>

<223> v-region + CH1 constant region gamma-1 with signal

sequence

<400> 7

Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala

1 5 10 15

Tyr Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro

20 25 30

Gly Gly Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Tyr Ser Ile Thr

35 40 45

Ser Gly Tyr Ser Trp Asn Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu

50 55 60

Glu Trp Val Ala Ser Ile Thr Tyr Asp Gly Ser Thr Asn Tyr Asn Pro

65 70 75 80

Ser Val Lys Gly Arg Ile Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

85 90 95

Phe Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

100 105 110

Tyr Cys Ala Arg Gly Ser His Tyr Phe Gly His Trp His Phe Ala Val

115 120 125

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly

130 135 140

Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly

145 150 155 160

Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val

165 170 175

Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe

180 185 190

Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val

195 200 205

Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val

210 215 220

Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys

225 230 235 240

Ser Cys

<210> 8

<211> 726

<212> DNA

<213> Artificial sequence

<220>

<223> v-region + CH1 constant region gamma-1 with signal

sequence

<400> 8

atgaagtggg tcaccttcat ctccctgctg tttctgttct ccagcgccta ctccgaagtg 60

cagttggtgg agtcgggtgg agggctggtg cagcctggcg gtagcctgag gctgtcctgt 120

gccgtgtccg gatactccat tacctccggc tactcgtgga actggatcag acaggctccc 180

ggaaagggac ttgagtgggt ggcgtccatc acctacgacg gctcaaccaa ctataacccg 240

tccgtgaagg gccgcatcac catttcgcgc gacgacagca agaatacttt ttacctccaa 300

atgaacagcc tgcgggccga agatactgcc gtgtactact gcgcgcgggg atcacattac 360

ttcgggcact ggcacttcgc cgtctgggga cagggcaccc tcgtcactgt ctcgagcgct 420

tctacaaagg gcccatcggt cttccccctg gcaccctcct ccaagagcac ctctgggggc 480

acagcggccc tgggctgcct ggtcaaggac tacttccccg aaccggtgac ggtgtcgtgg 540

aactcaggcg ccctgaccag cggcgtgcac accttcccgg ctgtcctaca gtcctcagga 600

ctctactccc tcagcagcgt ggtgaccgtg ccctccagca gcttgggcac ccagacctac 660

atctgcaacg tgaatcacaa gcccagcaac accaaggtgg acaagaaagt tgagcccaaa 720

tcttgt 726

<210> 9

<211> 451

<212> PROTEIN

<213> Artificial sequence

<220>

<223> v-region + full-length gamma-1 constant region

<400> 9

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Tyr Ser Ile Thr Ser Gly

20 25 30

Tyr Ser Trp Asn Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp

35 40 45

Val Ala Ser Ile Thr Tyr Asp Gly Ser Thr Asn Tyr Asn Pro Ser Val

50 55 60

Lys Gly Arg Ile Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Phe Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Ser His Tyr Phe Gly His Trp His Phe Ala Val Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

355 360 365

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Lys

450

<210> 10

<211> 1353

<212> DNA

<213> Artificial sequence

<220>

<223> v-region + full-length gamma-1 constant region

<400> 10

gaagtgcagt tggtggagtc gggtggaggg ctggtgcagc ctggcggtag cctgaggctg 60

tcctgtgccg tgtccggata ctccattacc tccggctact cgtggaactg gatcagacag 120

gctcccggaa agggacttga gtgggtggcg tccatcacct acgacggctc aaccaactat 180

aacccgtccg tgaagggccg catcaccatt tcgcgcgacg acagcaagaa tactttttac 240

ctccaaatga acagcctgcg ggccgaagat actgccgtgt actactgcgc gcggggatca 300

cattacttcg ggcactggca cttcgccgtc tggggacagg gcaccctcgt cactgtctcg 360

agcgcttcta caaagggccc ctccgtgttc ccgctcgctc catcatcgaa gtctaccagc 420

ggaggcactg cggctctcgg ttgcctcgtg aaggactact tcccggagcc ggtgaccgtg 480

tcgtggaaca gcggagccct gaccagcggg gtgcacacct ttccggccgt cttgcagtca 540

agcggccttt actccctgtc atcagtggtg actngtcccgt ccagctcatt gggaacccaa 600

acctacatct gcaatgtgaa tcacaaacct agcaacacca aggttgacaa gaaagtcgag 660

cccaaatcgt gtgacaagac tcacacttgt ccgccgtgcc cggcacccga actgctggga 720

ggtcccagcg tctttctgtt ccctccaaag ccgaaagaca cgctgatgat ctcccgcacc 780

ccggaggtca cttgcgtggt cgtggacgtg tcacatgagg acccagaggt gaagttcaat 840

tggtacgtgg atggcgtcga agtccacaat gccaaaacta agcccagaga agaacagtac 900

aattcgacct accgcgtcgt gtccgtgctc acggtgttgc atcaggattg gctgaacggg 960

aaggaataca agtgcaaagt gtccaacaag gcgctgccgg caccgatcga gaaaactatc 1020

tccaaagcga agggacagcc tagggaacct caagtctaca cgctgccacc atcacgggaa 1080

gaaatgacta agaatcaagt ctcactgact tgtctggtga aggggtttta ccctagcgac 1140

attgccgtgg agtgggaatc caacggccag ccagagaaca actacaagac tacccctcca 1200

gtgctcgact cggatggatc gttcttcctt tactcgaagc tcaccgtgga taagtcccgg 1260

tggcagcagg gaaacgtgtt ctcctgctcg gtgatgcatg aagccctcca taaccactat 1320

acccaaaagt cgctgtccct gtcgccggga aag 1353

<210> 11

<211> 469

<212> PROTEIN

<213> Artificial sequence

<220>

<223> v-region + full-length gamma-1 constant region with signal

sequence

<400> 11

Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala

1 5 10 15

Tyr Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro

20 25 30

Gly Gly Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Tyr Ser Ile Thr

35 40 45

Ser Gly Tyr Ser Trp Asn Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu

50 55 60

Glu Trp Val Ala Ser Ile Thr Tyr Asp Gly Ser Thr Asn Tyr Asn Pro

65 70 75 80

Ser Val Lys Gly Arg Ile Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

85 90 95

Phe Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

100 105 110

Tyr Cys Ala Arg Gly Ser His Tyr Phe Gly His Trp His Phe Ala Val

115 120 125

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly

130 135 140

Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly

145 150 155 160

Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val

165 170 175

Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe

180 185 190

Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val

195 200 205

Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val

210 215 220

Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys

225 230 235 240

Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu

245 250 255

Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

260 265 270

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

275 280 285

Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

290 295 300

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser

305 310 315 320

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

325 330 335

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala

340 345 350

Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

355 360 365

Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln

370 375 380

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

385 390 395 400

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

405 410 415

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

420 425 430

Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser

435 440 445

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

450 455 460

Leu Ser Pro Gly Lys

465

<210> 12

<211> 1407

<212> DNA

<213> Artificial sequence

<220>

<223> v-region + full-length gamma-1 constant region with signal

sequence

<400> 12

atgaagtggg tcaccttcat ctccctgctg tttctgttct ccagcgccta ctccgaagtg 60

cagttggtgg agtcgggtgg agggctggtg cagcctggcg gtagcctgag gctgtcctgt 120

gccgtgtccg gatactccat tacctccggc tactcgtgga actggatcag acaggctccc 180

ggaaagggac ttgagtgggt ggcgtccatc acctacgacg gctcaaccaa ctataacccg 240

tccgtgaagg gccgcatcac catttcgcgc gacgacagca agaatacttt ttacctccaa 300

atgaacagcc tgcgggccga agatactgcc gtgtactact gcgcgcgggg atcacattac 360

ttcgggcact ggcacttcgc cgtctgggga cagggcaccc tcgtcactgt ctcgagcgct 420

tctacaaagg gcccctccgt gttcccgctc gctccatcat cgaagtctac cagcggaggc 480

actgcggctc tcggttgcct cgtgaaggac tacttcccgg agccggtgac cgtgtcgtgg 540

aacagcggag ccctgaccag cggggtgcac acctttccgg ccgtcttgca gtcaagcggc 600

ctttactccc tgtcatcagt ggtgactgtc ccgtccagct cattgggaac ccaaacctac 660

atctgcaatg tgaatcacaa acctagcaac accaaggttg acaagaaagt cgagcccaaa 720

tcgtgtgaca agactcacac ttgtccgccg tgcccggcac ccgaactgct gggaggtccc 780

agcgtctttc tgttccctcc aaagccgaaa gacacgctga tgatctcccg caccccggag 840

gtcacttgcg tggtcgtgga cgtgtcacat gaggacccag aggtgaagtt caattggtac 900

gtggatggcg tcgaagtcca caatgccaaa actaagccca gagaagaaca gtacaattcg 960

acctaccgcg tcgtgtccgt gctcacggtg ttgcatcagg attggctgaa cgggaaggaa 1020

tacaagtgca aagtgtccaa caaggcgctg ccggcaccga tcgagaaaac tatctccaaa 1080

gcgaagggac agcctaggga acctcaagtc tacacgctgc caccatcacg ggaagaaatg 1140

actaagaatc aagtctcact gacttgtctg gtgaaggggt tttaccctag cgacattgcc 1200

gtggagtggg aatccaacgg ccagccagag aacaactaca agactacccc tccagtgctc 1260

gactcggatg gatcgttctt cctttactcg aagctcaccg tggataagtc ccggtggcag 1320

cagggaaacg tgttctcctg ctcggtgatg catgaagccc tccataacca ctatacccaa 1380

aagtcgctgt ccctgtcgcc gggaaag 1407

<210> 13

<211> 25

<212> PROTEIN

<213> Artificial sequence

<220>

<223>FR H1

<400> 13

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser

20 25

<210> 14

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223>CDRH1

<400> 14

Gly Tyr Ser Ile Thr Ser Gly Tyr Ser Trp Asn

1 5 10

<210> 15

<211> 14

<212> PROTEIN

<213> Artificial sequence

<220>

<223>FR H2

<400> 15

Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala

1 5 10

<210> 16

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<223>CDRH2

<400> 16

Ser Ile Thr Tyr Asp Gly Ser Thr Asn Tyr Asn Pro Ser Val Lys Gly

1 5 10 15

<210> 17

<211> 32

<212> PROTEIN

<213> Artificial sequence

<220>

<223>FR H3

<400> 17

Arg Ile Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Phe Tyr Leu Gln

1 5 10 15

Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg

20 25 30

<210> 18

<211> 12

<212> PROTEIN

<213> Artificial sequence

<220>

<223>CDRH3

<400> 18

Gly Ser His Tyr Phe Gly His Trp His Phe Ala Val

1 5 10

<210> 19

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223>FR H4

<400> 19

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

1 5 10

<210> 20

<211> 111

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Light chain V region

<400> 20

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Asp Tyr Asp

20 25 30

Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Ser Tyr Leu Glu Ser Gly Val Pro Ser

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser His

85 90 95

Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 21

<211> 333

<212> DNA

<213> Artificial sequence

<220>

<223> Light chain V region

<400> 21

gatattcagc tgactcagag cccgagctca ctctccgctt ccgtggggga tagagtgacc 60

atcacttgcc gggcatccca gtcggtggac tacgacggag actcctacat gaactggtat 120

cagcagaagc ccggaaaagc cccaaagttg ctgatctacg ccgcctcata ccttgaaagc 180

ggcgtgcctt cgcgcttctc gggaagcggg tcgggcaccg atttcaccct gaccatttcg 240

tcgctgcagc cggaggactt cgcgacttac tactgccaac agtcccacga ggacccctat 300

acgtttggcc agggaaccaa ggtcgaaatc aag 333

<210> 22

<211> 129

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Light chain V region with signal sequence

<400> 22

Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala

1 5 10 15

Tyr Ser Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser

20 25 30

Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Asp

35 40 45

Tyr Asp Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Lys

50 55 60

Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Tyr Leu Glu Ser Gly Val

65 70 75 80

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

85 90 95

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln

100 105 110

Ser His Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile

115 120 125

Lys

<210> 23

<211> 387

<212> DNA

<213> Artificial sequence

<220>

<223> Light chain V region with signal sequence

<400> 23

atgaagtggg tcaccttcat ctccctgctg tttctgttct cctccgccta ctccgatatt 60

cagctgactc agagcccgag ctcactctcc gcttccgtgg gggatagagt gaccatcact 120

tgccgggcat cccagtcggt ggactacgac ggagactcct acatgaactg gtatcagcag 180

aagcccggaa aagccccaaa gttgctgatc tacgccgcct cataccttga aagcggcgtg 240

ccttcgcgct tctcgggaag cgggtcgggc accgatttca ccctgaccat ttcgtcgctg 300

cagccggagg acttcgcgac ttactactgc caacagtccc acgaggaccc ctatacgttt 360

ggccagggaa ccaaggtcga aatcaag 387

<210> 24

<211> 218

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Light chain V region + kappa constant region

<400> 24

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Asp Tyr Asp

20 25 30

Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Ser Tyr Leu Glu Ser Gly Val Pro Ser

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser His

85 90 95

Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 25

<211> 654

<212> DNA

<213> Artificial sequence

<220>

<223> V-region + kappa constant region

<400> 25

gatattcagc tgactcagag cccgagctca ctctccgctt ccgtggggga tagagtgacc 60

atcacttgcc gggcatccca gtcggtggac tacgacggag actcctacat gaactggtat 120

cagcagaagc ccggaaaagc cccaaagttg ctgatctacg ccgcctcata ccttgaaagc 180

ggcgtgcctt cgcgcttctc gggaagcggg tcgggcaccg atttcaccct gaccatttcg 240

tcgctgcagc cggaggactt cgcgacttac tactgccaac agtcccacga ggacccctat 300

acgtttggcc agggaaccaa ggtcgaaatc aagcgtacgg tagcggcccc atctgtcttc 360

atcttcccgc catctgatga gcagttgaaa tctggaactg cctctgttgt gtgcctgctg 420

aataacttct atcccagaga ggccaaagta cagtggaagg tggataacgc cctccaatcg 480

ggtaactccc aggagagtgt cacagagcag gacagcaagg acagcaccta cagcctcagc 540

agcaccctga cgctgagcaa agcagactac gagaaacaca aagtctacgc ctgcgaagtc 600

acccatcagg gcctgagctc gcccgtcaca aagagcttca acaggggaga gtgt 654

<210> 26

<211> 236

<212> PROTEIN

<213> Artificial sequence

<220>

<223> V region + kappa constant region with signal sequence

<400> 26

Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala

1 5 10 15

Tyr Ser Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser

20 25 30

Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Asp

35 40 45

Tyr Asp Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Lys

50 55 60

Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Tyr Leu Glu Ser Gly Val

65 70 75 80

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

85 90 95

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln

100 105 110

Ser His Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile

115 120 125

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

130 135 140

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

145 150 155 160

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

165 170 175

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

180 185 190

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

195 200 205

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

210 215 220

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230 235

<210> 27

<211> 708

<212> DNA

<213> Artificial sequence

<220>

<223> V region + kappa constant region with signal sequence

<400> 27

atgaagtggg tcaccttcat ctccctgctg tttctgttct cctccgccta ctccgatatt 60

cagctgactc agagcccgag ctcactctcc gcttccgtgg gggatagagt gaccatcact 120

tgccgggcat cccagtcggt ggactacgac ggagactcct acatgaactg gtatcagcag 180

aagcccggaa aagccccaaa gttgctgatc tacgccgcct cataccttga aagcggcgtg 240

ccttcgcgct tctcgggaag cgggtcgggc accgatttca ccctgaccat ttcgtcgctg 300

cagccggagg acttcgcgac ttactactgc caacagtccc acgaggaccc ctatacgttt 360

ggccagggaa ccaaggtcga aatcaagcgt acggtagcgg ccccatctgt cttcatcttc 420

ccgccatctg atgagcagtt gaaatctgga actgcctctg ttgtgtgcct gctgaataac 480

ttctatccca gagaggccaa agtacagtgg aaggtggata acgccctcca atcgggtaac 540

tcccaggaga gtgtcacaga gcaggacagc aaggacagca cctacagcct cagcagcacc 600

ctgacgctga gcaaagcaga ctacgagaaa cacaaagtct acgcctgcga agtcacccat 660

cagggcctga gctcgcccgt cacaaagagc ttcaacaggg gagagtgt 708

<210> 28

<211> 23

<212> PROTEIN

<213> Artificial sequence

<220>

<223>FR L1

<400> 28

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys

20

<210> 29

<211> 15

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRL1

<400> 29

Arg Ala Ser Gln Ser Val Asp Tyr Asp Gly Asp Ser Tyr Met Asn

1 5 10 15

<210> 30

<211> 15

<212> PROTEIN

<213> Artificial sequence

<220>

<223>FR L2

<400> 30

Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

1 5 10 15

<210> 31

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRL2

<400> 31

Ala Ala Ser Tyr Leu Glu Ser

15

<210> 32

<211> 32

<212> PROTEIN

<213> Artificial sequence

<220>

<223>FR L3

<400> 32

Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

1 5 10 15

Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys

20 25 30

<210> 33

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRL3

<400> 33

Gln Gln Ser His Glu Asp Pro Tyr Thr

15

<210> 34

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223>FR L4

<400> 34

Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

1 5 10

<210> 35

<211> 111

<212> PROTEIN

<213> Artificial sequence

<220>

<223> S60M_S77R_Q79R_v-area

<400> 35

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Asp Tyr Asp

20 25 30

Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Ser Tyr Leu Glu Ser Gly Val Pro Met

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Arg Leu Arg Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser His

85 90 95

Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 36

<211> 333

<212> DNA

<213> Artificial sequence

<220>

<223> S60M_S77R_Q79R_v-area

<400> 36

gatattcagc tgactcagag cccgagctca ctctccgctt ccgtggggga tagagtgacc 60

atcacttgcc gggcatccca gtcggtggac tacgacggag actcctacat gaactggtat 120

cagcagaagc ccggaaaagc cccaaagttg ctgatctacg ccgcctcata ccttgaaagc 180

ggcgtgccta tgcgcttctc gggaagcggg tcgggcaccg atttcaccct gaccatttcg 240

agactgaggc cggaggactt cgcgacttac tactgccaac agtcccacga ggacccctat 300

acgtttggcc agggaaccaa ggtcgaaatc aag 333

<210> 37

<211> 129

<212> PROTEIN

<213> Artificial sequence

<220>

<223> S60M_S77R_Q79R_v-region with signal sequence

<400> 37

Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala

1 5 10 15

Tyr Ser Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser

20 25 30

Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Asp

35 40 45

Tyr Asp Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Lys

50 55 60

Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Tyr Leu Glu Ser Gly Val

65 70 75 80

Pro Met Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

85 90 95

Ile Ser Arg Leu Arg Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln

100 105 110

Ser His Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile

115 120 125

Lys

<210> 38

<211> 387

<212> DNA

<213> Artificial sequence

<220>

<223> S60M_S77R_Q79R_v-region with signal sequence

<400> 38

atgaagtggg tcaccttcat ctccctgctg tttctgttct cctccgccta ctccgatatt 60

cagctgactc agagcccgag ctcactctcc gcttccgtgg gggatagagt gaccatcact 120

tgccgggcat cccagtcggt ggactacgac ggagactcct acatgaactg gtatcagcag 180

aagcccggaa aagccccaaa gttgctgatc tacgccgcct cataccttga aagcggcgtg 240

cctatgcgct tctcgggaag cgggtcgggc accgatttca ccctgaccat ttcgagactg 300

aggccggagg acttcgcgac ttactactgc caacagtccc acgaggaccc ctatacgttt 360

ggccagggaa ccaaggtcga aatcaag 387

<210> 39

<211> 218

<212> PROTEIN

<213> Artificial sequence

<220>

<223> S60M_S77R_Q79R_v-region + kappa constant region including L154P

<400> 39

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Asp Tyr Asp

20 25 30

Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Ser Tyr Leu Glu Ser Gly Val Pro Met

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Arg Leu Arg Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser His

85 90 95

Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Pro Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 40

<211> 654

<212> DNA

<213> Artificial sequence

<220>

<223> S60M_S77R_Q79R_v-region + kappa constant region including L154P

<400> 40

gatattcagc tgactcagag cccgagctca ctctccgctt ccgtggggga tagagtgacc 60

atcacttgcc gggcatccca gtcggtggac tacgacggag actcctacat gaactggtat 120

cagcagaagc ccggaaaagc cccaaagttg ctgatctacg ccgcctcata ccttgaaagc 180

ggcgtgccta tgcgcttctc gggaagcggg tcgggcaccg atttcaccct gaccatttcg 240

agactgaggc cggaggactt cgcgacttac tactgccaac agtcccacga ggacccctat 300

acgtttggcc agggaaccaa ggtcgaaatc aagcgtacgg tagcggcccc atctgtcttc 360

atcttcccgc catctgatga gcagttgaaa tctggaactg cctctgttgt gtgcctgctg 420

aataacttct atcccagaga ggccaaagta cagtggaagg tggataacgc cccgcaatcg 480

ggtaactccc aggagagtgt cacagagcag gacagcaagg acagcaccta cagcctcagc 540

agcaccctga cgctgagcaa agcagactac gagaaacaca aagtctacgc ctgcgaagtc 600

acccatcagg gcctgagctc gcccgtcaca aagagcttca acaggggaga gtgt 654

<210> 41

<211> 236

<212> PROTEIN

<213> Artificial sequence

<220>

<223> S60M_S77R_Q79R_v-region + kappa constant region including L154P,

with signal sequence

<400> 41

Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala

1 5 10 15

Tyr Ser Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser

20 25 30

Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Asp

35 40 45

Tyr Asp Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Lys

50 55 60

Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Tyr Leu Glu Ser Gly Val

65 70 75 80

Pro Met Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

85 90 95

Ile Ser Arg Leu Arg Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln

100 105 110

Ser His Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile

115 120 125

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

130 135 140

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

145 150 155 160

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Pro

165 170 175

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

180 185 190

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

195 200 205

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

210 215 220

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230 235

<210> 42

<211> 708

<212> DNA

<213> Artificial sequence

<220>

<223> S60M_S77R_Q79R_v-region + kappa constant region including L154P,

with signal sequence

<400> 42

atgaagtggg tcaccttcat ctccctgctg tttctgttct cctccgccta ctccgatatt 60

cagctgactc agagcccgag ctcactctcc gcttccgtgg gggatagagt gaccatcact 120

tgccgggcat cccagtcggt ggactacgac ggagactcct acatgaactg gtatcagcag 180

aagcccggaa aagccccaaa gttgctgatc tacgccgcct cataccttga aagcggcgtg 240

cctatgcgct tctcgggaag cgggtcgggc accgatttca ccctgaccat ttcgagactg 300

aggccggagg acttcgcgac ttactactgc caacagtccc acgaggaccc ctatacgttt 360

ggccagggaa ccaaggtcga aatcaagcgt acggtagcgg ccccatctgt cttcatcttc 420

ccgccatctg atgagcagtt gaaatctgga actgcctctg ttgtgtgcct gctgaataac 480

ttctatccca gagaggccaa agtacagtgg aaggtggata acgccccgca atcgggtaac 540

tcccaggaga gtgtcacaga gcaggacagc aaggacagca cctacagcct cagcagcacc 600

ctgacgctga gcaaagcaga ctacgagaaa cacaaagtct acgcctgcga agtcacccat 660

cagggcctga gctcgcccgt cacaaagagc ttcaacaggg gagagtgt 708

<210> 43

<211> 32

<212> PROTEIN

<213> Artificial sequence

<220>

<223>FR L3 S64M S60M

<400> 43

Gly Val Pro Met Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

1 5 10 15

Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys

20 25 30

<210> 44

<211> 32

<212> PROTEIN

<213> Artificial sequence

<220>

<223>FR L3 S81R S77R

<400> 44

Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

1 5 10 15

Leu Thr Ile Ser Arg Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys

20 25 30

<210> 45

<211> 32

<212> PROTEIN

<213> Artificial sequence

<220>

<223>FR L3 Q83R Q79R

<400> 45

Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

1 5 10 15

Leu Thr Ile Ser Ser Leu Arg Pro Glu Asp Phe Ala Thr Tyr Tyr Cys

20 25 30

<210> 46

<211> 32

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FR L3 S64M S81R S60M S77R

<400> 46

Gly Val Pro Met Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

1 5 10 15

Leu Thr Ile Ser Arg Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys

20 25 30

<210> 47

<211> 32

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FR L3 S64M Q83R S60M Q79R

<400> 47

Gly Val Pro Met Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

1 5 10 15

Leu Thr Ile Ser Ser Leu Arg Pro Glu Asp Phe Ala Thr Tyr Tyr Cys

20 25 30

<210> 48

<211> 32

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FR L3 S81R Q83R S77R Q79R

<400> 48

Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

1 5 10 15

Leu Thr Ile Ser Arg Leu Arg Pro Glu Asp Phe Ala Thr Tyr Tyr Cys

20 25 30

<210> 49

<211> 32

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FR L3 S64M S81R Q83R S60M S77R Q79R

<400> 49

Gly Val Pro Met Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

1 5 10 15

Leu Thr Ile Ser Arg Leu Arg Pro Glu Asp Phe Ala Thr Tyr Tyr Cys

20 25 30

<210> 50

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223>CDRL2 S56D S52D

<400> 50

Ala Ala Asp Tyr Leu Glu Ser

15

<210> 51

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CDRL2 S56E S52E

<400> 51

Ala Ala Glu Tyr Leu Glu Ser

15

<210> 52

<211> 32

<212> PROTEIN

<213> Artificial sequence

<220>

<223>FR L3 S71M S67M

<400> 52

Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

1 5 10 15

Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys

20 25 30

<210> 53

<211> 32

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FR L3 S64M S71M S60M S67M

<400> 53

Gly Val Pro Met Arg Phe Ser Gly Ser Gly Met Gly Thr Asp Phe Thr

1 5 10 15

Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys

20 25 30

<210> 54

<211> 32

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FR L3 S81R S71M S77R S67M

<400> 54

Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Met Gly Thr Asp Phe Thr

1 5 10 15

Leu Thr Ile Ser Arg Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys

20 25 30

<210> 55

<211> 32

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FR L3 Q83R S71M Q79R S67M

<400> 55

Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Met Gly Thr Asp Phe Thr

1 5 10 15

Leu Thr Ile Ser Ser Leu Arg Pro Glu Asp Phe Ala Thr Tyr Tyr Cys

20 25 30

<210> 56

<211> 32

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FR L3 S64M S81R S71M S60M S77R S67M

<400> 56

Gly Val Pro Met Arg Phe Ser Gly Ser Gly Met Gly Thr Asp Phe Thr

1 5 10 15

Leu Thr Ile Ser Arg Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys

20 25 30

<210> 57

<211> 32

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FR L3 S64M Q83R S71M S60M Q79R S67M

<400> 57

Gly Val Pro Met Arg Phe Ser Gly Ser Gly Met Gly Thr Asp Phe Thr

1 5 10 15

Leu Thr Ile Ser Ser Leu Arg Pro Glu Asp Phe Ala Thr Tyr Tyr Cys

20 25 30

<210> 58

<211> 32

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FR L3 S81R Q83R S71M S77R Q79R S67M

<400> 58

Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Met Gly Thr Asp Phe Thr

1 5 10 15

Leu Thr Ile Ser Arg Leu Arg Pro Glu Asp Phe Ala Thr Tyr Tyr Cys

20 25 30

<210> 59

<211> 32

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FR L3 S64M S81R Q83R S71M S60M S77R Q79R S67M

<400> 59

Gly Val Pro Met Arg Phe Ser Gly Ser Gly Met Gly Thr Asp Phe Thr

1 5 10 15

Leu Thr Ile Ser Arg Leu Arg Pro Glu Asp Phe Ala Thr Tyr Tyr Cys

20 25 30

<210> 60

<211> 32

<212> PROTEIN

<213> Artificial sequence

<220>

<223>FR L3 S67Y S63Y

<400> 60

Gly Val Pro Ser Arg Phe Tyr Gly Ser Gly Ser Gly Thr Asp Phe Thr

1 5 10 15

Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys

20 25 30

<210> 61

<211> 32

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FR L3 S64M S67Y S60M S63Y

<400> 61

Gly Val Pro Met Arg Phe Tyr Gly Ser Gly Ser Gly Thr Asp Phe Thr

1 5 10 15

Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys

20 25 30

<210> 62

<211> 32

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FR L3 S81R S67Y S77R S63Y

<400> 62

Gly Val Pro Ser Arg Phe Tyr Gly Ser Gly Ser Gly Thr Asp Phe Thr

1 5 10 15

Leu Thr Ile Ser Arg Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys

20 25 30

<210> 63

<211> 32

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FR L3 Q83R S67Y Q79R S63Y

<400> 63

Gly Val Pro Ser Arg Phe Tyr Gly Ser Gly Ser Gly Thr Asp Phe Thr

1 5 10 15

Leu Thr Ile Ser Ser Leu Arg Pro Glu Asp Phe Ala Thr Tyr Tyr Cys

20 25 30

<210> 64

<211> 32

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FR L3 S64M S81R S67Y S60M S77R S63Y

<400> 64

Gly Val Pro Met Arg Phe Tyr Gly Ser Gly Ser Gly Thr Asp Phe Thr

1 5 10 15

Leu Thr Ile Ser Arg Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys

20 25 30

<210> 65

<211> 32

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FR L3 S64M Q83R S67Y S60M Q79R S63Y

<400> 65

Gly Val Pro Met Arg Phe Tyr Gly Ser Gly Ser Gly Thr Asp Phe Thr

1 5 10 15

Leu Thr Ile Ser Ser Leu Arg Pro Glu Asp Phe Ala Thr Tyr Tyr Cys

20 25 30

<210> 66

<211> 32

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FR L3 S81R Q83R S67Y S77R Q79R S63Y

<400> 66

Gly Val Pro Ser Arg Phe Tyr Gly Ser Gly Ser Gly Thr Asp Phe Thr

1 5 10 15

Leu Thr Ile Ser Arg Leu Arg Pro Glu Asp Phe Ala Thr Tyr Tyr Cys

20 25 30

<210> 67

<211> 32

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FR L3 S64M S81R Q83R S67Y S60M S77R Q79R S63Y

<400> 67

Gly Val Pro Met Arg Phe Tyr Gly Ser Gly Ser Gly Thr Asp Phe Thr

1 5 10 15

Leu Thr Ile Ser Arg Leu Arg Pro Glu Asp Phe Ala Thr Tyr Tyr Cys

20 25 30

<210> 68

<211> 32

<212> PROTEIN

<213> Artificial sequence

<220>

<223>FR L3 S80N S76N

<400> 68

Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

1 5 10 15

Leu Thr Ile Asn Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys

20 25 30

<210> 69

<211> 32

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FR L3 S64M S80N S60M S76N

<400> 69

Gly Val Pro Met Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

1 5 10 15

Leu Thr Ile Asn Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys

20 25 30

<210> 70

<211> 32

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FR L3 S81R S80N S77R S76

<400> 70

Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

1 5 10 15

Leu Thr Ile Asn Arg Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys

20 25 30

<210> 71

<211> 32

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FR L3 Q83R S80N Q79R S76N

<400> 71

Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

1 5 10 15

Leu Thr Ile Asn Ser Leu Arg Pro Glu Asp Phe Ala Thr Tyr Tyr Cys

20 25 30

<210> 72

<211> 32

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FR L3 S64M S81R S80N S60M S77R S76N

<400> 72

Gly Val Pro Met Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

1 5 10 15

Leu Thr Ile Asn Arg Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys

20 25 30

<210> 73

<211> 32

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FR L3 S64M Q83R S80N S60M Q79R S76N

<400> 73

Gly Val Pro Met Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

1 5 10 15

Leu Thr Ile Asn Ser Leu Arg Pro Glu Asp Phe Ala Thr Tyr Tyr Cys

20 25 30

<210> 74

<211> 32

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FR L3 S81R Q83R S80N S77R Q79R S76N

<400> 74

Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

1 5 10 15

Leu Thr Ile Asn Arg Leu Arg Pro Glu Asp Phe Ala Thr Tyr Tyr Cys

20 25 30

<210> 75

<211> 32

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FR L3 S64M S81R Q83R S80N S60M S77R Q79R S76N

<400> 75

Gly Val Pro Met Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

1 5 10 15

Leu Thr Ile Asn Arg Leu Arg Pro Glu Asp Phe Ala Thr Tyr Tyr Cys

20 25 30

<210> 76

<211> 32

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FR L3 S80N S67Y S76N S63Y

<400> 76

Gly Val Pro Ser Arg Phe Tyr Gly Ser Gly Ser Gly Thr Asp Phe Thr

1 5 10 15

Leu Thr Ile Asn Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys

20 25 30

<210> 77

<211> 32

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FR L3 S64M S80N S67Y S60M S76N S63Y

<400> 77

Gly Val Pro Met Arg Phe Tyr Gly Ser Gly Ser Gly Thr Asp Phe Thr

1 5 10 15

Leu Thr Ile Asn Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys

20 25 30

<210> 78

<211> 32

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FR L3 S81R S80N S67Y S77R S76N S63Y

<400> 78

Gly Val Pro Ser Arg Phe Tyr Gly Ser Gly Ser Gly Thr Asp Phe Thr

1 5 10 15

Leu Thr Ile Asn Arg Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys

20 25 30

<210> 79

<211> 32

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FR L3 Q83R S80N S67Y Q79R S76N S63Y

<400> 79

Gly Val Pro Ser Arg Phe Tyr Gly Ser Gly Ser Gly Thr Asp Phe Thr

1 5 10 15

Leu Thr Ile Asn Ser Leu Arg Pro Glu Asp Phe Ala Thr Tyr Tyr Cys

20 25 30

<210> 80

<211> 32

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FR L3 S64M S81R S80N S67Y S60M S77R S76N S63Y

<400> 80

Gly Val Pro Met Arg Phe Tyr Gly Ser Gly Ser Gly Thr Asp Phe Thr

1 5 10 15

Leu Thr Ile Asn Arg Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys

20 25 30

<210> 81

<211> 32

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FR L3 S64M Q83R S80N S67Y S60M Q79R S76N S63Y

<400> 81

Gly Val Pro Met Arg Phe Tyr Gly Ser Gly Ser Gly Thr Asp Phe Thr

1 5 10 15

Leu Thr Ile Asn Ser Leu Arg Pro Glu Asp Phe Ala Thr Tyr Tyr Cys

20 25 30

<210> 82

<211> 32

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FR L3 S81R Q83R S80N S67Y S77R Q79R S76N S63Y

<400> 82

Gly Val Pro Ser Arg Phe Tyr Gly Ser Gly Ser Gly Thr Asp Phe Thr

1 5 10 15

Leu Thr Ile Asn Arg Leu Arg Pro Glu Asp Phe Ala Thr Tyr Tyr Cys

20 25 30

<210> 83

<211> 32

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FR L3 S64M S81R Q83R S80N S67Y S60M S77R Q79R S76N S63Y

<400> 83

Gly Val Pro Met Arg Phe Tyr Gly Ser Gly Ser Gly Thr Asp Phe Thr

1 5 10 15

Leu Thr Ile Asn Arg Leu Arg Pro Glu Asp Phe Ala Thr Tyr Tyr Cys

20 25 30

<210> 84

<211> 32

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FR L3 S67Y S71M S63Y S67M

<400> 84

Gly Val Pro Ser Arg Phe Tyr Gly Ser Gly Met Gly Thr Asp Phe Thr

1 5 10 15

Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys

20 25 30

<210> 85

<211> 32

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FR L3 S64M S67Y S71M S60M S63Y S67M

<400> 85

Gly Val Pro Met Arg Phe Tyr Gly Ser Gly Met Gly Thr Asp Phe Thr

1 5 10 15

Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys

20 25 30

<210> 86

<211> 32

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FR L3 S81R S67Y S71M S77R S63Y S67M

<400> 86

Gly Val Pro Ser Arg Phe Tyr Gly Ser Gly Met Gly Thr Asp Phe Thr

1 5 10 15

Leu Thr Ile Ser Arg Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys

20 25 30

<210> 87

<211> 32

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FR L3 Q83R S67Y S71M Q79R S63Y S67M

<400> 87

Gly Val Pro Ser Arg Phe Tyr Gly Ser Gly Met Gly Thr Asp Phe Thr

1 5 10 15

Leu Thr Ile Ser Ser Leu Arg Pro Glu Asp Phe Ala Thr Tyr Tyr Cys

20 25 30

<210> 88

<211> 32

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FR L3 S64M S81R S67Y S71M S60M S77R S63Y S67M

<400> 88

Gly Val Pro Met Arg Phe Tyr Gly Ser Gly Met Gly Thr Asp Phe Thr

1 5 10 15

Leu Thr Ile Ser Arg Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys

20 25 30

<210> 89

<211> 32

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FR L3 S64M Q83R S67Y S71M S60M Q79R S63Y S67M

<400> 89

Gly Val Pro Met Arg Phe Tyr Gly Ser Gly Met Gly Thr Asp Phe Thr

1 5 10 15

Leu Thr Ile Ser Ser Leu Arg Pro Glu Asp Phe Ala Thr Tyr Tyr Cys

20 25 30

<210> 90

<211> 32

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FR L3 S81R Q83R S67Y S71M S77R Q79R S63Y S67M

<400> 90

Gly Val Pro Ser Arg Phe Tyr Gly Ser Gly Met Gly Thr Asp Phe Thr

1 5 10 15

Leu Thr Ile Ser Arg Leu Arg Pro Glu Asp Phe Ala Thr Tyr Tyr Cys

20 25 30

<210> 91

<211> 32

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FR L3 S64M S81R Q83R S67Y S71M S60M S77R Q79R S63Y S67M

<400> 91

Gly Val Pro Met Arg Phe Tyr Gly Ser Gly Met Gly Thr Asp Phe Thr

1 5 10 15

Leu Thr Ile Ser Arg Leu Arg Pro Glu Asp Phe Ala Thr Tyr Tyr Cys

20 25 30

<210> 92

<211> 32

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FR L3 S80N S71M S76N S67M

<400> 92

Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Met Gly Thr Asp Phe Thr

1 5 10 15

Leu Thr Ile Asn Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys

20 25 30

<210> 93

<211> 32

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FR L3 S64M S80N S71M S60M S76N S67M (

<400> 93

Gly Val Pro Met Arg Phe Ser Gly Ser Gly Met Gly Thr Asp Phe Thr

1 5 10 15

Leu Thr Ile Asn Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys

20 25 30

<210> 94

<211> 32

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FR L3 S81R S80N S71M S77R S76N S67M

<400> 94

Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Met Gly Thr Asp Phe Thr

1 5 10 15

Leu Thr Ile Asn Arg Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys

20 25 30

<210> 95

<211> 32

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FR L3 Q83R S80N S71M Q79R S76N S67M

<400> 95

Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Met Gly Thr Asp Phe Thr

1 5 10 15

Leu Thr Ile Asn Ser Leu Arg Pro Glu Asp Phe Ala Thr Tyr Tyr Cys

20 25 30

<210> 96

<211> 32

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FR L3 S64M S81R S80N S71M S60M S77R S76N S67M

<400> 96

Gly Val Pro Met Arg Phe Ser Gly Ser Gly Met Gly Thr Asp Phe Thr

1 5 10 15

Leu Thr Ile Asn Arg Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys

20 25 30

<210> 97

<211> 32

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FR L3 S64M Q83R S80N S71M S60M Q79R S76N S67M

<400> 97

Gly Val Pro Met Arg Phe Ser Gly Ser Gly Met Gly Thr Asp Phe Thr

1 5 10 15

Leu Thr Ile Asn Ser Leu Arg Pro Glu Asp Phe Ala Thr Tyr Tyr Cys

20 25 30

<210> 98

<211> 32

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FR L3 S81R Q83R S80N S71M S77R Q79R S76N S67M

<400> 98

Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Met Gly Thr Asp Phe Thr

1 5 10 15

Leu Thr Ile Asn Arg Leu Arg Pro Glu Asp Phe Ala Thr Tyr Tyr Cys

20 25 30

<210> 99

<211> 32

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FR L3 S64M S81R Q83R S80N S71M S60M S77R Q79R S76N S67M

<400> 99

Gly Val Pro Met Arg Phe Ser Gly Ser Gly Met Gly Thr Asp Phe Thr

1 5 10 15

Leu Thr Ile Asn Arg Leu Arg Pro Glu Asp Phe Ala Thr Tyr Tyr Cys

20 25 30

<210> 100

<211> 32

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FR L3 S80N S67Y S71M S76N S63Y S67M

<400> 100

Gly Val Pro Ser Arg Phe Tyr Gly Ser Gly Met Gly Thr Asp Phe Thr

1 5 10 15

Leu Thr Ile Asn Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys

20 25 30

<210> 101

<211> 32

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FR L3 S64M S80N S67Y S71M S60M S76N S63Y S67M

<400> 101

Gly Val Pro Met Arg Phe Tyr Gly Ser Gly Met Gly Thr Asp Phe Thr

1 5 10 15

Leu Thr Ile Asn Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys

20 25 30

<210> 102

<211> 32

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FR L3 S81R S80N S67Y S71M S77R S76N S63Y S67M

<400> 102

Gly Val Pro Ser Arg Phe Tyr Gly Ser Gly Met Gly Thr Asp Phe Thr

1 5 10 15

Leu Thr Ile Asn Arg Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys

20 25 30

<210> 103

<211> 32

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FR L3 Q83R S80N S67Y S71M Q79R S76N S63Y S67M

<400> 103

Gly Val Pro Ser Arg Phe Tyr Gly Ser Gly Met Gly Thr Asp Phe Thr

1 5 10 15

Leu Thr Ile Asn Ser Leu Arg Pro Glu Asp Phe Ala Thr Tyr Tyr Cys

20 25 30

<210> 104

<211> 32

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FR L3 S64M S81R S80N S67Y S71M S60M S77R S76N S63Y S67M

<400> 104

Gly Val Pro Met Arg Phe Tyr Gly Ser Gly Met Gly Thr Asp Phe Thr

1 5 10 15

Leu Thr Ile Asn Arg Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys

20 25 30

<210> 105

<211> 32

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FR L3 S64M Q83R S80N S67Y S71M S60M Q79R S76N S63Y S67M

<400> 105

Gly Val Pro Met Arg Phe Tyr Gly Ser Gly Met Gly Thr Asp Phe Thr

1 5 10 15

Leu Thr Ile Asn Ser Leu Arg Pro Glu Asp Phe Ala Thr Tyr Tyr Cys

20 25 30

<210> 106

<211> 32

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FR L3 S81R Q83R S80N S67Y S71M S77R Q79R S76N S63Y S67M

<400> 106

Gly Val Pro Ser Arg Phe Tyr Gly Ser Gly Met Gly Thr Asp Phe Thr

1 5 10 15

Leu Thr Ile Asn Arg Leu Arg Pro Glu Asp Phe Ala Thr Tyr Tyr Cys

20 25 30

<210> 107

<211> 30

<212> PROTEIN

<213> Artificial sequence

<220>

<223> FR L3 S64M S81R Q83R S80N S67Y S71M S60M S77R Q79R S76N S63Y

S67M

<400> 107

Pro Met Arg Phe Tyr Gly Ser Gly Met Gly Thr Asp Phe Thr Leu Thr

1 5 10 15

Ile Asn Arg Leu Arg Pro Glu Asp Phe Ala Thr Tyr Tyr Cys

20 25 30

<210> 108

<211> 325

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Wild type human IgE-Fc (domains CE2-CE4 numbered V224-K547

according to Dorrington & Bennich (1978)

<400> 108

Val Ala Ser Arg Asp Phe Thr Pro Pro Thr Val Lys Ile Leu Gln Ser

1 5 10 15

Ser Cys Asp Gly Gly Gly His Phe Pro Pro Thr Ile Gln Leu Leu Cys

20 25 30

Leu Val Ser Gly Tyr Thr Pro Gly Thr Ile Asn Ile Thr Trp Leu Glu

35 40 45

Asp Gly Gln Val Met Asp Val Asp Leu Ser Thr Ala Ser Thr Thr Gln

50 55 60

Glu Gly Glu Leu Ala Ser Thr Gln Ser Glu Leu Thr Leu Ser Gln Lys

65 70 75 80

His Trp Leu Ser Asp Arg Thr Tyr Thr Cys Gln Val Thr Tyr Gln Gly

85 90 95

His Thr Phe Glu Asp Ser Thr Lys Lys Cys Ala Asp Ser Asn Pro Arg

100 105 110

Gly Val Ser Ala Tyr Leu Ser Arg Pro Ser Pro Phe Asp Leu Phe Ile

115 120 125

Arg Lys Ser Pro Thr Ile Thr Cys Leu Val Val Asp Leu Ala Pro Ser

130 135 140

Lys Gly Thr Val Asn Leu Thr Trp Ser Arg Ala Ser Gly Lys Pro Val

145 150 155 160

Asn His Ser Thr Arg Lys Glu Glu Lys Gln Arg Asn Gly Thr Leu Thr

165 170 175

Val Thr Ser Thr Leu Pro Val Gly Thr Arg Asp Trp Ile Glu Gly Glu

180 185 190

Thr Tyr Gln Cys Arg Val Thr His Pro His Leu Pro Arg Ala Leu Met

195 200 205

Arg Ser Thr Thr Lys Thr Ser Gly Pro Arg Ala Ala Pro Glu Val Tyr

210 215 220

Ala Phe Ala Thr Pro Glu Trp Pro Gly Ser Arg Asp Lys Arg Thr Leu

225 230 235 240

Ala Cys Leu Ile Gln Asn Phe Met Pro Glu Asp Ile Ser Val Gln Trp

245 250 255

Leu His Asn Glu Val Gln Leu Pro Asp Ala Arg His Ser Thr Thr Gln

260 265 270

Pro Arg Lys Thr Lys Gly Ser Gly Phe Phe Val Phe Ser Arg Leu Glu

275 280 285

Val Thr Arg Ala Glu Trp Glu Gln Lys Asp Glu Phe Ile Cys Arg Ala

290 295 300

Val His Glu Ala Ala Ser Pro Ser Gln Thr Val Gln Arg Ala Val Ser

305 310 315 320

Val Asn Pro Gly Lys

325

<210> 109

<211> 111

<212> PROTEIN

<213> Artificial sequence

<220>

<223> S77R_Q79R_v-area (Fab1)

<400> 109

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Asp Tyr Asp

20 25 30

Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Ser Tyr Leu Glu Ser Gly Val Pro Ser

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Arg Leu Arg Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser His

85 90 95

Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 110

<211> 333

<212> DNA

<213> Artificial sequence

<220>

<223> 77R_Q79R_v-area (Fab1)

<400> 110

gatattcagc tgactcagag cccgagctca ctctccgctt ccgtggggga tagagtgacc 60

atcacttgcc gggcatccca gtcggtggac tacgacggag actcctacat gaactggtac 120

cagcagaagc ccggaaaagc cccaaagttg ctgatctacg ccgcctccta ccttgaaagc 180

ggcgtgcctt cacgcttctc gggaagcggg tctggcaccg atttcaccct gaccatttcg 240

agactgaggc cggaggactt cgcgacttac tactgccaac agtcccacga ggacccctat 300

acgtttggcc agggtaccaa ggtcgaaatc aag 333

<210> 111

<211> 129

<212> PROTEIN

<213> Artificial sequence

<220>

<223> S77R_Q79R_v-region (Fab1) with signal sequence

<400> 111

Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala

1 5 10 15

Tyr Ser Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser

20 25 30

Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Asp

35 40 45

Tyr Asp Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Lys

50 55 60

Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Tyr Leu Glu Ser Gly Val

65 70 75 80

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

85 90 95

Ile Ser Arg Leu Arg Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln

100 105 110

Ser His Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile

115 120 125

Lys

<210> 112

<211> 387

<212> DNA

<213> Artificial sequence

<220>

<223> S77R_Q79R_v-region (Fab1) with signal sequence

<400> 112

atgaagtggg tcaccttcat ctccctgctg tttctgttct cctccgccta ctccgatatt 60

cagctgactc agagcccgag ctcactctcc gcttccgtgg gggatagagt gaccatcact 120

tgccgggcat cccagtcggt ggactacgac ggagactcct acatgaactg gtaccagcag 180

aagcccggaa aagccccaaa gttgctgatc tacgccgcct cctaccttga aagcggcgtg 240

ccttcacgct tctcgggaag cgggtctggc accgatttca ccctgaccat ttcgagactg 300

aggccggagg acttcgcgac ttactactgc caacagtccc acgaggaccc ctatacgttt 360

ggccagggta ccaaggtcga aatcaag 387

<210> 113

<211> 111

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Fab2_v-region

<400> 113

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Asp Tyr Asp

20 25 30

Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Ser Tyr Leu Glu Ser Gly Val Pro Ser

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser His

85 90 95

Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 114

<211> 333

<212> DNA

<213> Artificial sequence

<220>

<223> Fab2_v-region

<400> 114

gatattcagc tgactcagag cccgagctca ctctccgctt ccgtggggga tagagtgacc 60

atcacttgcc gggcatccca gtcggtggac tacgacggag actcctacat gaactggtat 120

cagcagaagc ccggaaaagc cccaaagttg ctgatctacg ccgcctcata ccttgaaagc 180

ggcgtgcctt cgcgcttctc gggaagcggg tcgggcaccg atttcaccct gaccatttcg 240

tcgctgcagc cggaggactt cgcgacttac tactgccaac agtcccacga ggacccctat 300

acgtttggcc agggaaccaa ggtcgaaatc aag 333

<210> 115

<211> 129

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Fab2_v-region with signal sequence

<400> 115

Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala

1 5 10 15

Tyr Ser Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser

20 25 30

Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Asp

35 40 45

Tyr Asp Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Lys

50 55 60

Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Tyr Leu Glu Ser Gly Val

65 70 75 80

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

85 90 95

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln

100 105 110

Ser His Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile

115 120 125

Lys

<210> 116

<211> 387

<212> DNA

<213> Artificial sequence

<220>

<223> Fab2_v-region with signal sequence

<400> 116

atgaagtggg tcaccttcat ctccctgctg tttctgttct cctccgccta ctccgatatt 60

cagctgactc agagcccgag ctcactctcc gcttccgtgg gggatagagt gaccatcact 120

tgccgggcat cccagtcggt ggactacgac ggagactcct acatgaactg gtatcagcag 180

aagcccggaa aagccccaaa gttgctgatc tacgccgcct cataccttga aagcggcgtg 240

ccttcgcgct tctcgggaag cgggtcgggc accgatttca ccctgaccat ttcgtcgctg 300

cagccggagg acttcgcgac ttactactgc caacagtccc acgaggaccc ctatacgttt 360

ggccagggaa ccaaggtcga aatcaag 387

<210> 117

<211> 218

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Fab2_v region + kappa constant region including L154P

<400> 117

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Asp Tyr Asp

20 25 30

Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Ser Tyr Leu Glu Ser Gly Val Pro Ser

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser His

85 90 95

Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Pro Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 118

<211> 654

<212> DNA

<213> Artificial sequence

<220>

<223> Fab2_v region + kappa constant region including L154P

<400> 118

gatattcagc tgactcagag cccgagctca ctctccgctt ccgtggggga tagagtgacc 60

atcacttgcc gggcatccca gtcggtggac tacgacggag actcctacat gaactggtat 120

cagcagaagc ccggaaaagc cccaaagttg ctgatctacg ccgcctcata ccttgaaagc 180

ggcgtgcctt cgcgcttctc gggaagcggg tcgggcaccg atttcaccct gaccatttcg 240

tcgctgcagc cggaggactt cgcgacttac tactgccaac agtcccacga ggacccctat 300

acgtttggcc agggaaccaa ggtcgaaatc aagcgtacgg tagcggcccc atctgtcttc 360

atcttcccgc catctgatga gcagttgaaa tctggaactg cctctgttgt gtgcctgctg 420

aataacttct atcccagaga ggccaaagta cagtggaagg tggataacgc cccgcaatcg 480

ggtaactccc aggagagtgt cacagagcag gacagcaagg acagcaccta cagcctcagc 540

agcaccctga cgctgagcaa agcagactac gagaaacaca aagtctacgc ctgcgaagtc 600

acccatcagg gcctgagctc gcccgtcaca aagagcttca acaggggaga gtgt 654

<210> 119

<211> 236

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Fab2_v region + kappa constant region including L154P,

with signal sequence

<400> 119

Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala

1 5 10 15

Tyr Ser Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser

20 25 30

Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Asp

35 40 45

Tyr Asp Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Lys

50 55 60

Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Tyr Leu Glu Ser Gly Val

65 70 75 80

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

85 90 95

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln

100 105 110

Ser His Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile

115 120 125

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

130 135 140

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

145 150 155 160

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Pro

165 170 175

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

180 185 190

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

195 200 205

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

210 215 220

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230 235

<210> 120

<211> 708

<212> DNA

<213> Artificial sequence

<220>

<223> Fab2_v region + kappa constant region including L154P,

with signal sequence

<400> 120

atgaagtggg tcaccttcat ctccctgctg tttctgttct cctccgccta ctccgatatt 60

cagctgactc agagcccgag ctcactctcc gcttccgtgg gggatagagt gaccatcact 120

tgccgggcat cccagtcggt ggactacgac ggagactcct acatgaactg gtatcagcag 180

aagcccggaa aagccccaaa gttgctgatc tacgccgcct cataccttga aagcggcgtg 240

ccttcgcgct tctcgggaag cgggtcgggc accgatttca ccctgaccat ttcgtcgctg 300

cagccggagg acttcgcgac ttactactgc caacagtccc acgaggaccc ctatacgttt 360

ggccagggaa ccaaggtcga aatcaagcgt acggtagcgg ccccatctgt cttcatcttc 420

ccgccatctg atgagcagtt gaaatctgga actgcctctg ttgtgtgcct gctgaataac 480

ttctatccca gagaggccaa agtacagtgg aaggtggata acgccccgca atcgggtaac 540

tcccaggaga gtgtcacaga gcaggacagc aaggacagca cctacagcct cagcagcacc 600

ctgacgctga gcaaagcaga ctacgagaaa cacaaagtct acgcctgcga agtcacccat 660

cagggcctga gctcgcccgt cacaaagagc ttcaacaggg gagagtgt 708

<210> 121

<211> 111

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Fab3_S77R_Q79R_v-area

<400> 121

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Asp Tyr Asp

20 25 30

Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Ser Tyr Leu Glu Ser Gly Val Pro Ser

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Arg Leu Arg Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser His

85 90 95

Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 122

<211> 333

<212> DNA

<213> Artificial sequence

<220>

<223> Fab3_S77R_Q79R_v-area

<400> 122

gatattcagc tgactcagag cccgagctca ctctccgctt ccgtggggga tagagtgacc 60

atcacttgcc gggcatccca gtcggtggac tacgacggag actcctacat gaactggtac 120

cagcagaagc ccggaaaagc cccaaagttg ctgatctacg ccgcctccta ccttgaaagc 180

ggcgtgcctt cacgcttctc gggaagcggg tctggcaccg atttcaccct gaccatttcg 240

agactgaggc cggaggactt cgcgacttac tactgccaac agtcccacga ggacccctat 300

acgtttggcc agggtaccaa ggtcgaaatc aag 333

<210> 123

<211> 129

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Fab3_S77R_Q79R_v-region with signal sequence

<400> 123

Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala

1 5 10 15

Tyr Ser Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser

20 25 30

Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Asp

35 40 45

Tyr Asp Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Lys

50 55 60

Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Tyr Leu Glu Ser Gly Val

65 70 75 80

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

85 90 95

Ile Ser Arg Leu Arg Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln

100 105 110

Ser His Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile

115 120 125

Lys

<210> 124

<211> 387

<212> DNA

<213> Artificial sequence

<220>

<223> Fab3_S77R_Q79R_v-region with signal sequence

<400> 124

atgaagtggg tcaccttcat ctccctgctg tttctgttct cctccgccta ctccgatatt 60

cagctgactc agagcccgag ctcactctcc gcttccgtgg gggatagagt gaccatcact 120

tgccgggcat cccagtcggt ggactacgac ggagactcct acatgaactg gtaccagcag 180

aagcccggaa aagccccaaa gttgctgatc tacgccgcct cctaccttga aagcggcgtg 240

ccttcacgct tctcgggaag cgggtctggc accgatttca ccctgaccat ttcgagactg 300

aggccggagg acttcgcgac ttactactgc caacagtccc acgaggaccc ctatacgttt 360

ggccagggta ccaaggtcga aatcaag 387

<210> 125

<211> 218

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Fab3_S77R_Q79R_v-region + kappa constant region including L154P

<400> 125

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Asp Tyr Asp

20 25 30

Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Ser Tyr Leu Glu Ser Gly Val Pro Ser

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Arg Leu Arg Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser His

85 90 95

Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Pro Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 126

<211> 654

<212> DNA

<213> Artificial sequence

<220>

<223> Fab3_S77R_Q79R_v-region + kappa constant region including L154P

<400> 126

gatattcagc tgactcagag cccgagctca ctctccgctt ccgtggggga tagagtgacc 60

atcacttgcc gggcatccca gtcggtggac tacgacggag actcctacat gaactggtac 120

cagcagaagc ccggaaaagc cccaaagttg ctgatctacg ccgcctccta ccttgaaagc 180

ggcgtgcctt cacgcttctc gggaagcggg tctggcaccg atttcaccct gaccatttcg 240

agactgaggc cggaggactt cgcgacttac tactgccaac agtcccacga ggacccctat 300

acgtttggcc agggtaccaa ggtcgaaatc aagcgtacgg tagcggcccc atctgtcttc 360

atcttcccgc catctgatga gcagttgaaa tctggaactg cctctgttgt gtgcctgctg 420

aataacttct atcccagaga ggccaaagta cagtggaagg tggataacgc cccgcaatcg 480

ggtaactccc aggagagtgt cacagagcag gacagcaagg acagcaccta cagcctcagc 540

agcaccctga cgctgagcaa agcagactac gagaaacaca aagtctacgc ctgcgaagtc 600

acccatcagg gcctgagctc gcccgtcaca aagagcttca acaggggaga gtgt 654

<210> 127

<211> 236

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Fab3_S77R_Q79R_v-region + kappa constant region including L154P,

with signal sequence

<400> 127

Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala

1 5 10 15

Tyr Ser Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser

20 25 30

Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Asp

35 40 45

Tyr Asp Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Lys

50 55 60

Ala Pro Lys Leu Leu Ile Tyr Ala Ala Ser Tyr Leu Glu Ser Gly Val

65 70 75 80

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

85 90 95

Ile Ser Arg Leu Arg Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln

100 105 110

Ser His Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile

115 120 125

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

130 135 140

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

145 150 155 160

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Pro

165 170 175

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

180 185 190

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

195 200 205

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

210 215 220

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

225 230 235

<210> 128

<211> 708

<212> DNA

<213> Artificial sequence

<220>

<223> Fab3_S77R_Q79R_v-region + kappa constant region including L154P,

with signal sequence

<400> 128

atgaagtggg tcaccttcat ctccctgctg tttctgttct cctccgccta ctccgatatt 60

cagctgactc agagcccgag ctcactctcc gcttccgtgg gggatagagt gaccatcact 120

tgccgggcat cccagtcggt ggactacgac ggagactcct acatgaactg gtaccagcag 180

aagcccggaa aagccccaaa gttgctgatc tacgccgcct cctaccttga aagcggcgtg 240

ccttcacgct tctcgggaag cgggtctggc accgatttca ccctgaccat ttcgagactg 300

aggccggagg acttcgcgac ttactactgc caacagtccc acgaggaccc ctatacgttt 360

ggccagggta ccaaggtcga aatcaagcgt acggtagcgg ccccatctgt cttcatcttc 420

ccgccatctg atgagcagtt gaaatctgga actgcctctg ttgtgtgcct gctgaataac 480

ttctatccca gagaggccaa agtacagtgg aaggtggata acgccccgca atcgggtaac 540

tcccaggaga gtgtcacaga gcaggacagc aaggacagca cctacagcct cagcagcacc 600

ctgacgctga gcaaagcaga ctacgagaaa cacaaagtct acgcctgcga agtcacccat 660

cagggcctga gctcgcccgt cacaaagagc ttcaacaggg gagagtgt 708

<210> 129

<211> 107

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Omalizumab_v-region

<400> 129

Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr

1 5 10 15

Ile Thr Cys Arg Ala Ser Gln Ser Val Asp Tyr Asp Gly Asp Ser Tyr

20 25 30

Met Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Tyr Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser His Glu Asp Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 130

<211> 321

<212> DNA

<213> Artificial sequence

<220>

<223> Omalizumab_v-region

<400> 130

actcagagcc cgagctcact ctccgcttcc gtgggggata gagtgaccat cacttgccgg 60

gcatcccagt cggtggacta cgacggagac tcctacatga actggtatca gcagaagccc 120

ggaaaagccc caaagttgct gatctacgcc gcctcatacc ttgaaagcgg cgtgccttcg 180

cgcttctcgg gaagcgggtc gggcaccgat ttcaccctga ccatttcgtc gctgcagccg 240

gaggacttcg cgacttacta ctgccaacag tcccacgagg acccctatac gtttggccag 300

ggaaccaagg tcgaaatcaa g 321

<210> 131

<211> 32

<212> PROTEIN

<213> Artificial sequence

<220>

<223>FR3

<400> 131

Gly Val Pro Met Arg Phe Ser Gly Ser Gly Met Gly Thr Asp Phe Thr

1 5 10 15

Leu Thr Ile Ser Arg Leu Arg Pro Glu Asp Phe Ala Thr Tyr Tyr Cys

20 25 30

<210> 132

<211> 111

<212> PROTEIN

<213> Artificial sequence

<220>

<223> S60M_S52D_S67M_S77R_Q79R_v-area

<400> 132

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Asp Tyr Asp

20 25 30

Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Asp Tyr Leu Glu Ser Gly Val Pro Met

50 55 60

Arg Phe Ser Gly Ser Gly Met Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Arg Leu Arg Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser His

85 90 95

Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 133

<211> 333

<212> DNA

<213> Artificial sequence

<220>

<223> S60M_S52D_S67M_S77R_Q79R_v-area

<400> 133

gatattcagc tgactcagag cccgagctca ctctccgctt ccgtggggga tagagtgacc 60

atcacttgcc gggcatccca gtcggtggac tacgacggag actcctacat gaactggtat 120

cagcagaagc ccggaaaagc cccaaagttg ctgatctacg ccgccgacta ccttgaaagc 180

ggcgtgccta tgcgcttctc gggaagcggg atgggcaccg atttcaccct gaccatttcg 240

agactgaggc cggaggactt cgcgacttac tactgccaac agtcccacga ggacccctat 300

acgtttggcc agggaaccaa ggtcgaaatc aag 333

<210> 134

<211> 129

<212> PROTEIN

<213> Artificial sequence

<220>

<223> S60M_S52D_S67M_S77R_Q79R_v-region with signal sequence

<400> 134

Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala

1 5 10 15

Tyr Ser Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser

20 25 30

Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Asp

35 40 45

Tyr Asp Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Lys

50 55 60

Ala Pro Lys Leu Leu Ile Tyr Ala Ala Asp Tyr Leu Glu Ser Gly Val

65 70 75 80

Pro Met Arg Phe Ser Gly Ser Gly Met Gly Thr Asp Phe Thr Leu Thr

85 90 95

Ile Ser Arg Leu Arg Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln

100 105 110

Ser His Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile

115 120 125

Lys

<210> 135

<211> 387

<212> DNA

<213> Artificial sequence

<220>

<223> S60M_S52D_S67M_S77R_Q79R_v-region with signal sequence

<400> 135

atgaagtggg tcaccttcat ctccctgctg tttctgttct cctccgccta ctccgatatt 60

cagctgactc agagcccgag ctcactctcc gcttccgtgg gggatagagt gaccatcact 120

tgccgggcat cccagtcggt ggactacgac ggagactcct acatgaactg gtatcagcag 180

aagcccggaa aagccccaaa gttgctgatc tacgccgccg actaccttga aagcggcgtg 240

cctatgcgct tctcgggaag cgggatgggc accgatttca ccctgaccat ttcgagactg 300

aggccggagg acttcgcgac ttactactgc caacagtccc acgaggaccc ctatacgttt 360

ggccagggaa ccaaggtcga aatcaag 387

<210> 136

<211> 218

<212> PROTEIN

<213> Artificial sequence

<220>

<223> S60M_S52D_S67M_S77R_Q79R_v-region + kappa constant region

<400> 136

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Asp Tyr Asp

20 25 30

Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Asp Tyr Leu Glu Ser Gly Val Pro Met

50 55 60

Arg Phe Ser Gly Ser Gly Met Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Arg Leu Arg Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser His

85 90 95

Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 137

<211> 654

<212> DNA

<213> Artificial sequence

<220>

<223> S60M_S52D_S67M_S77R_Q79R_v-region + kappa constant region

<400> 137

gatattcagc tgactcagag cccgagctca ctctccgctt ccgtggggga tagagtgacc 60

atcacttgcc gggcatccca gtcggtggac tacgacggag actcctacat gaactggtat 120

cagcagaagc ccggaaaagc cccaaagttg ctgatctacg ccgccgacta ccttgaaagc 180

ggcgtgccta tgcgcttctc gggaagcggg atgggcaccg atttcaccct gaccatttcg 240

agactgaggc cggaggactt cgcgacttac tactgccaac agtcccacga ggacccctat 300

acgtttggcc agggaaccaa ggtcgaaatc aagcgtacgg tagcggcccc atctgtcttc 360

atcttcccgc catctgatga gcagttgaaa tctggaactg cctctgttgt gtgcctgctg 420

aataacttct atcccagaga ggccaaagta cagtggaagg tggataacgc cctccaatcg 480

ggtaactccc aggagagtgt cacagagcag gacagcaagg acagcaccta cagcctcagc 540

agcaccctga cgctgagcaa agcagactac gagaaacaca aagtctacgc ctgcgaagtc 600

acccatcagg gcctgagctc gcccgtcaca aagagcttca acaggggaga gtgt 654

<210> 138

<211> 32

<212> PROTEIN

<213> Artificial sequence

<220>

<223>FR3

<400> 138

Gly Val Pro Arg Arg Phe Ser Gly Ser Gly Met Gly Thr Asp Phe Thr

1 5 10 15

Leu Thr Ile Ser Arg Leu Arg Pro Glu Asp Phe Ala Thr Tyr Tyr Cys

20 25 30

<210> 139

<211> 111

<212> PROTEIN

<213> Artificial sequence

<220>

<223> S60R_S52D_S67M_S77R_Q79R_v-area

<400> 139

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Asp Tyr Asp

20 25 30

Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Asp Tyr Leu Glu Ser Gly Val Pro Arg

50 55 60

Arg Phe Ser Gly Ser Gly Met Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Arg Leu Arg Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser His

85 90 95

Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 140

<211> 333

<212> DNA

<213> Artificial sequence

<220>

<223> S60R_S52D_S67M_S77R_Q79R_v-area

<400> 140

gatattcagc tgactcagag cccgagctca ctctccgctt ccgtggggga tagagtgacc 60

atcacttgcc gggcatccca gtcggtggac tacgacggag actcctacat gaactggtat 120

cagcagaagc ccggaaaagc cccaaagttg ctgatctacg ccgccgatta ccttgaaagc 180

ggcgtgcctc gtcgcttctc gggaagcggg atgggcaccg atttcaccct gaccatttcg 240

agactgaggc cggaggactt cgcgacttac tactgccaac agtcccacga ggacccctat 300

acgtttggcc agggaaccaa ggtcgaaatc aag 333

<210> 141

<211> 129

<212> PROTEIN

<213> Artificial sequence

<220>

<223> S60R_S52D_S67M_S77R_Q79R_v-region with signal sequence

<400> 141

Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala

1 5 10 15

Tyr Ser Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser

20 25 30

Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Asp

35 40 45

Tyr Asp Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Lys

50 55 60

Ala Pro Lys Leu Leu Ile Tyr Ala Ala Asp Tyr Leu Glu Ser Gly Val

65 70 75 80

Pro Arg Arg Phe Ser Gly Ser Gly Met Gly Thr Asp Phe Thr Leu Thr

85 90 95

Ile Ser Arg Leu Arg Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln

100 105 110

Ser His Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile

115 120 125

Lys

<210> 142

<211> 387

<212> DNA

<213> Artificial sequence

<220>

<223> S60R_S52D_S67M_S77R_Q79R_v-region with signal sequence

<400> 142

atgaagtggg tcaccttcat ctccctgctg tttctgttct cctccgccta ctccgatatt 60

cagctgactc agagcccgag ctcactctcc gcttccgtgg gggatagagt gaccatcact 120

tgccgggcat cccagtcggt ggactacgac ggagactcct acatgaactg gtatcagcag 180

aagcccggaa aagccccaaa gttgctgatc tacgccgccg attaccttga aagcggcgtg 240

cctcgtcgct tctcgggaag cgggatgggc accgatttca ccctgaccat ttcgagactg 300

aggccggagg acttcgcgac ttactactgc caacagtccc acgaggaccc ctatacgttt 360

ggccagggaa ccaaggtcga aatcaag 387

<210> 143

<211> 218

<212> PROTEIN

<213> Artificial sequence

<220>

<223> S60R_S52D_S67M_S77R_Q79R_v-region + kappa constant region

<400> 143

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Asp Tyr Asp

20 25 30

Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Asp Tyr Leu Glu Ser Gly Val Pro Arg

50 55 60

Arg Phe Ser Gly Ser Gly Met Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Arg Leu Arg Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser His

85 90 95

Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 144

<211> 654

<212> DNA

<213> Artificial sequence

<220>

<223> S60R_S52D_S67M_S77R_Q79R_v-region + kappa constant region

<400> 144

gatattcagc tgactcagag cccgagctca ctctccgctt ccgtggggga tagagtgacc 60

atcacttgcc gggcatccca gtcggtggac tacgacggag actcctacat gaactggtat 120

cagcagaagc ccggaaaagc cccaaagttg ctgatctacg ccgccgatta ccttgaaagc 180

ggcgtgcctc gtcgcttctc gggaagcggg atgggcaccg atttcaccct gaccatttcg 240

agactgaggc cggaggactt cgcgacttac tactgccaac agtcccacga ggacccctat 300

acgtttggcc agggaaccaa ggtcgaaatc aagcgtacgg tagcggcccc atctgtcttc 360

atcttcccgc catctgatga gcagttgaaa tctggaactg cctctgttgt gtgcctgctg 420

aataacttct atcccagaga ggccaaagta cagtggaagg tggataacgc cctccaatcg 480

ggtaactccc aggagagtgt cacagagcag gacagcaagg acagcaccta cagcctcagc 540

agcaccctga cgctgagcaa agcagactac gagaaacaca aagtctacgc ctgcgaagtc 600

acccatcagg gcctgagctc gcccgtcaca aagagcttca acaggggaga gtgt 654

<210> 145

<211> 111

<212> PROTEIN

<213> Artificial sequence

<220>

<223> S60K_S52D_S67M_S77R_Q79R_v-area

<400> 145

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Asp Tyr Asp

20 25 30

Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Asp Tyr Leu Glu Ser Gly Val Pro Lys

50 55 60

Arg Phe Ser Gly Ser Gly Met Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Arg Leu Arg Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser His

85 90 95

Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 146

<211> 111

<212> PROTEIN

<213> Artificial sequence

<220>

<223> S60Q_S52D_S67M_S77R_Q79R_v-area

<400> 146

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Asp Tyr Asp

20 25 30

Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Asp Tyr Leu Glu Ser Gly Val Pro Gln

50 55 60

Arg Phe Ser Gly Ser Gly Met Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Arg Leu Arg Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser His

85 90 95

Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 147

<211> 506

<212> PROTEIN

<213> Artificial sequence

<220>

<223> v-region + CH1 gamma-1 constant region plus linker plus CA645

gL4gH5 scFv with signal sequence

<400> 147

Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala

1 5 10 15

Tyr Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro

20 25 30

Gly Gly Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Tyr Ser Ile Thr

35 40 45

Ser Gly Tyr Ser Trp Asn Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu

50 55 60

Glu Trp Val Ala Ser Ile Thr Tyr Asp Gly Ser Thr Asn Tyr Asn Pro

65 70 75 80

Ser Val Lys Gly Arg Ile Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr

85 90 95

Phe Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

100 105 110

Tyr Cys Ala Arg Gly Ser His Tyr Phe Gly His Trp His Phe Ala Val

115 120 125

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly

130 135 140

Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly

145 150 155 160

Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val

165 170 175

Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe

180 185 190

Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val

195 200 205

Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val

210 215 220

Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys

225 230 235 240

Ser Cys Ser Gly Gly Gly Gly Thr Gly Gly Gly Gly Ser Glu Val Gln

245 250 255

Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg

260 265 270

Leu Ser Cys Ala Val Ser Gly Ile Asp Leu Ser Asn Tyr Ala Ile Asn

275 280 285

Trp Val Arg Gln Ala Pro Gly Lys Cys Leu Glu Trp Ile Gly Ile Ile

290 295 300

Trp Ala Ser Gly Thr Thr Phe Tyr Ala Thr Trp Ala Lys Gly Arg Phe

305 310 315 320

Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu Gln Met Asn

325 330 335

Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Thr Val

340 345 350

Pro Gly Tyr Ser Thr Ala Pro Tyr Phe Asp Leu Trp Gly Gln Gly Thr

355 360 365

Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

370 375 380

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln

385 390 395 400

Ser Pro Ser Ser Val Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr

405 410 415

Cys Gln Ser Ser Pro Ser Val Trp Ser Asn Phe Leu Ser Trp Tyr Gln

420 425 430

Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Glu Ala Ser Lys

435 440 445

Leu Thr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr

450 455 460

Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr

465 470 475 480

Tyr Tyr Cys Gly Gly Gly Tyr Ser Ser Ile Ser Asp Thr Thr Phe Gly

485 490 495

Cys Gly Thr Lys Val Glu Ile Lys Arg Thr

500 505

<210> 148

<211> 1518

<212> DNA

<213> Artificial sequence

<220>

<223> v-region + CH1 gamma-1 constant region plus linker plus CA645

gL4gH5 scFv with signal sequence

<400> 148

atgaagtggg tcaccttcat ctccctgctg tttctgttct ccagcgccta ctccgaagtg 60

cagttggtgg agtcgggtgg agggctggtg cagcctggcg gtagcctgag gctgtcctgt 120

gccgtgtccg gatactccat tacctccggc tactcgtgga actggatcag acaggctccc 180

ggaaagggac ttgagtgggt ggcgtccatc acctacgacg gctcaaccaa ctataacccg 240

tccgtgaagg gccgcatcac catttcgcgc gacgacagca agaatacttt ttacctccaa 300

atgaacagcc tgcgggccga agatactgcc gtgtactact gcgcgcgggg atcacattac 360

ttcgggcact ggcacttcgc cgtctgggga cagggcaccc tcgtcactgt ctcgagcgct 420

tctacaaagg gcccatcggt cttccccctg gcaccctcct ccaagagcac ctctgggggc 480

acagcggccc tgggctgcct ggtcaaggac tacttccccg aaccagtgac ggtgtcgtgg 540

aactcaggtg ccctgaccag cggcgttcac accttcccgg ctgtcctaca gtcttcagga 600

ctctactccc tgagcagcgt ggtgaccgtg ccctccagca gcttgggcac ccagacctac 660

atctgcaacg tgaatcacaa gcccagcaac accaaggtcg ataagaaagt tgagcccaaa 720

tcttgtagtg gaggtggggg caccggtgga ggtggcagcg aggttcaact gcttgagtct 780

ggaggaggcc tagtccagcc tggagggagc ctgcgtctct cttgtgcagt aagcggcatc 840

gacctgagca attacgccat caactgggtg agacaagctc cggggaagtg tttagaatgg 900

atcggtataa tatgggccag tgggacgacc ttttatgcta catgggcgaa aggaaggttt 960

acaattagcc gggacaatag caaaaacacc gtgtatctcc aaatgaactc cttgcgagca 1020

gaggacacgg cggtgtacta ttgtgctcgc actgtcccag gttatagcac tgcaccctac 1080

ttcgatctgt ggggacaagg gaccctggtg actgtttcaa gtggcggagg gggtagtgga 1140

ggggggtggct ctggggggtgg cggaagcggt ggcggggggtt ctgacataca aatgactcag 1200

tctccttcat cggtatccgc gtccgttggc gatagggtga ctattacatg tcaaagctct 1260

cctagcgtct ggagcaattt tctatcctgg tatcaacaga aaccggggaa ggctccaaaa 1320

cttctgattt atgaagcctc gaaactcacc agtggagttc cgtcaagatt cagtggctct 1380

ggatcaggga cagacttcac gttgacaatc agttcgctgc aaccagagga ctttgcgacc 1440

tactattgtg gtggaggtta cagtagcata agtgatacga catttgggtg cggtactaag 1500

gtggaaatca aacgtacc 1518

<210> 149

<211> 11

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Linker between CH1 and CA645 gL4gH5 scFv

<400> 149

Ser Gly Gly Gly Gly Thr Gly Gly Gly Gly Ser

1 5 10

<210> 150

<211> 253

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CA645 gL4gH5 scFv

<400> 150

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Ile Asp Leu Ser Asn Tyr

20 25 30

Ala Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Cys Leu Glu Trp Ile

35 40 45

Gly Ile Ile Trp Ala Ser Gly Thr Thr Phe Tyr Ala Thr Trp Ala Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Thr Val Pro Gly Tyr Ser Thr Ala Pro Tyr Phe Asp Leu Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly

115 120 125

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln

130 135 140

Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly Asp Arg Val

145 150 155 160

Thr Ile Thr Cys Gln Ser Ser Pro Ser Val Trp Ser Asn Phe Leu Ser

165 170 175

Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Glu

180 185 190

Ala Ser Lys Leu Thr Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly

195 200 205

Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp

210 215 220

Phe Ala Thr Tyr Tyr Cys Gly Gly Gly Tyr Ser Ser Ile Ser Asp Thr

225 230 235 240

Thr Phe Gly Cys Gly Thr Lys Val Glu Ile Lys Arg Thr

245 250

<210> 151

<211> 20

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Linker between CA645 gH5 with CA645 gL4 in scFv

<400> 151

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser

20

<210> 152

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CA645 CDRH1

<400> 152

Gly Ile Asp Leu Ser Asn Tyr Ala Ile Asn

1 5 10

<210> 153

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CA645 CDRH2

<400> 153

Ile Ile Trp Ala Ser Gly Thr Thr Phe Tyr Ala Thr Trp Ala Lys Gly

1 5 10 15

<210> 154

<211> 13

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CA645 CDRH3

<400> 154

Thr Val Pro Gly Tyr Ser Thr Ala Pro Tyr Phe Asp Leu

1 5 10

<210> 155

<211> 12

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CA645 CDRL1

<400> 155

Gln Ser Ser Pro Ser Val Trp Ser Asn Phe Leu Ser

1 5 10

<210> 156

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CA645 CDRL2

<400> 156

Glu Ala Ser Lys Leu Thr Ser

15

<210> 157

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<223> CA645 CDRL3

<400> 157

Gly Gly Tyr Ser Ser Ile Ser Asp Thr Thr

1 5 10

<210> 158

<211> 111

<212> PROTEIN

<213> Artificial sequence

<220>

<223> S60M_S52D_S67M_S77R_Q79R_S63W_S76N_v-area

<400> 158

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Asp Tyr Asp

20 25 30

Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Asp Tyr Leu Glu Ser Gly Val Pro Met

50 55 60

Arg Phe Trp Gly Ser Gly Met Gly Thr Asp Phe Thr Leu Thr Ile Asn

65 70 75 80

Arg Leu Arg Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser His

85 90 95

Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 159

<211> 111

<212> PROTEIN

<213> Artificial sequence

<220>

<223> S60M_S52D_S67M_S77R_Q79R_S63Y_S76N_v-area

<400> 159

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Asp Tyr Asp

20 25 30

Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Asp Tyr Leu Glu Ser Gly Val Pro Met

50 55 60

Arg Phe Tyr Gly Ser Gly Met Gly Thr Asp Phe Thr Leu Thr Ile Asn

65 70 75 80

Arg Leu Arg Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser His

85 90 95

Glu Asp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 160

<211> 18

<212> PROTEIN

<213> Artificial sequence

<220>

<223> Signal sequence

<400> 160

Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala

1 5 10 15

Tyr Ser

<---

Claims (38)

1. Anti-IgE antibody, or antigen-binding agent, containing heavy chain variable region containing complementarity determining region, CDR-H1, with amino acid sequence SEQ ID NO:14, CDR-H2 with amino acid sequence SEQ ID NO:16, CDR-H3, with amino acid sequence SEQ ID NO:18, And light chain variable region containing a complementarity determining region, CDR-L1, with the amino acid sequence of SEQ ID NO:29, CDR-L2, with the amino acid sequence of SEQ ID NO:50 or SEQ ID NO:51, CDR-L3, with the amino acid sequence of SEQ ID NO:33; wherein the light chain variable region also comprises a framework region, FR-L3, with an amino acid sequence, SEQ ID NO:32, which has one, two, three, four, five, six, seven or more amino acid substitutions to enhance the interaction of the anti-IgE antibody , or antigen-binding agent, with the Cε2 domain of human IgE; and where: a. the FR-L3 region has a mutation at position S60 (Kabat) to M, R, K, N, Q or T; b. the FR-L3 region has a mutation at position S67 (Kabat) on M; c. the FR-L3 region has a mutation at position S77 (Kabat) to R; d. the FR-L3 region has a mutation at position Q79 (Kabat) to R. 2. Anti-IgE antibody according to claim 1, wherein the scFv contains a heavy chain variable region and a light chain variable region, preferably linked through a linker to the sequence SEQ ID NO:151, where the heavy chain variable region contains CDR-H1 with amino acid sequence SEQ ID NO:152, CDR-H2 with the amino acid sequence SEQ ID NO:153 and CDR-H3 with the amino acid sequence SEQ ID NO:154, And the light chain variable region contains CDR-L1 with the amino acid sequence SEQ ID NO:155, CDR-L2 with the amino acid sequence SEQ ID NO:156, CDR-L3 with the amino acid sequence SEQ ID NO:157. 3. Anti-IgE antibody according to claim 1 or 2, where the Fab fragment contains a heavy chain variable region and a light chain variable region, where: a. the heavy chain variable region contains CDR-H1 having the amino acid sequence of SEQ ID NO:14, CDR-H2 having the amino acid sequence of SEQ ID NO:16 and CDR-H3 having the amino acid sequence of SEQ ID NO:18, and the light chain variable region contains CDR-L1 with amino acid sequence SEQ ID NO:29, CDR-L2 with amino acid sequence SEQ ID NO:50, CDR-L3 with amino acid sequence SEQ ID NO:33 and framework region FW-L3 with amino acid sequence SEQ ID NO:131 or 138; or b. the heavy chain variable region contains the amino acid sequence of SEQ ID NO:1 and the light chain variable region contains an amino acid sequence selected from SEQ ID NO:132 or 139. 4. An anti-IgE antibody, or antigen-binding agent, which is selected from the group consisting of: Fab fragment, modified Fab' fragment, Fab' fragment, F(ab') ₂ fragment, Fv, scFv, scab, diabody , bispecific antibody, tribody, FabFv, Fab-Fv-Fv, tribody or (Fab-Fv) ² -Fc. 5. The anti-IgE antibody of claim 1, which is a Fab fragment linked directly or via a linker to an scFv that binds to a serum carrier protein such as human serum albumin. 6. An isolated DNA molecule encoding the heavy and/or light chain(s) of an anti-IgE antibody, or antigen-binding agent, according to claims 1-5. 7. A cloning vector for cloning an antibody according to claim 1, containing one or more DNA sequences selected from: SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40 or SEQ ID NO:42, or SEQ ID NO :133, or SEQ ID NO:135, or SEQ ID NO:137, or SEQ ID NO:140, or SEQ ID NO:142, or SEQ ID NO:144; wherein, optionally, the cloning vector also contains one or more DNA sequences selected from: SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10 or SEQ ID NO :12, or SEQ ID NO:148. 8. An expression vector for expressing antibodies according to claim 1, containing one or more DNA sequences selected from: SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40 or SEQ ID NO:42, or SEQ ID NO :133, or SEQ ID NO:135, or SEQ ID NO:137, or SEQ ID NO:140, or SEQ ID NO:142, or SEQ ID NO:144; wherein, optionally, the expression vector also contains one or more DNA sequences selected from: SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10 or SEQ ID NO :12, or SEQ ID NO:148. 9. Pharmaceutical composition for the treatment or prevention of one or more diseases: allergies; allergic asthma; severe asthma; moderate asthma; chronic spontaneous urticaria; chronic idiopathic urticaria; prolonged allergic rhinitis; seasonal allergic rhinitis; exacerbations of asthma; acute bronchospasm; status asthmaticus; hyper-IgE syndrome; allergic bronchopulmonary aspergillosis; prevention of anaphylactic reactions; food allergies; atopic dermatitis; allergic rhinitis; allergies to bee venom; idiopathic anaphylaxis; peanut allergies; allergies to latex; inflammatory skin diseases; urticaria (caused by sunlight, cold, localized heat and/or prolonged pressure); cutaneous mastocytosis; systemic mastocytosis; eosinophil-related gastrointestinal disease; bullous pemphigoid; interstitial cystitis; nasal polyps; idiopathic angioedema or non-allergic asthma, wherein the antibody comprises an anti-IgE antibody or antigen binding agent according to claims 1 to 5, in combination with one or more pharmaceutically acceptable excipients, diluents or carriers. 10. Use of an anti-IgE antibody or antigen-binding agent according to claims 1 to 5 as a medicament for the treatment of a disease that is ameliorated by modulating the biological activity of human IgE. 11. The use of an anti-IgE antibody, or an antigen-binding agent, according to claims 1-5 for the treatment or prevention of one or more diseases: allergies; allergic asthma; severe asthma; moderate asthma; chronic spontaneous urticaria; chronic idiopathic urticaria; prolonged allergic rhinitis; seasonal allergic rhinitis; exacerbations of asthma; acute bronchospasm; status asthmaticus; hyper-IgE syndrome; allergic bronchopulmonary aspergillosis; prevention of anaphylactic reactions; food allergies; atopic dermatitis; allergic rhinitis; allergies to bee venom; idiopathic anaphylaxis; peanut allergies; allergies to latex; inflammatory skin diseases; urticaria (caused by sunlight, cold, local heat and/or prolonged pressure); cutaneous mastocytosis; systemic mastocytosis; eosinophil-related gastrointestinal disease; bullous pemphigoid; interstitial cystitis; nasal polyps; idiopathic angioedema or non-allergic asthma. 12. Use of the composition according to claim 9 for the treatment or prevention of one or more diseases: allergies; allergic asthma; severe asthma; moderate asthma; chronic spontaneous urticaria; chronic idiopathic urticaria; prolonged allergic rhinitis; seasonal allergic rhinitis; exacerbations of asthma; acute bronchospasm; status asthmaticus; hyper-IgE syndrome; allergic bronchopulmonary aspergillosis; prevention of anaphylactic reactions; food allergies; atopic dermatitis; allergic rhinitis; allergies to bee venom; idiopathic anaphylaxis; peanut allergies; allergies to latex; inflammatory skin diseases; urticaria (caused by sunlight, cold, localized heat and/or prolonged pressure); cutaneous mastocytosis; systemic mastocytosis; eosinophil-related gastrointestinal disease; bullous pemphigoid; interstitial cystitis; nasal polyps; idiopathic angioedema or non-allergic asthma.

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