JP6068582B2 - Anti-CεmX antibody capable of binding to human mIgE on B lymphocytes - Google Patents

Description

  IgE plays a central role in the induction of type I hypersensitivity reactions that cause allergic asthma, allergic rhinitis, and atopic dermatitis. An allergic reaction is a response of the immune system to less harmful environmental substances such as house dust mites, tree and grass pollen, certain foods and drugs, and fire ant bites. In such a response, binding of the allergen to IgE on the surface of basophils and mast cells is a type I IgE.Fc receptor, which is a cross-link of IgE and the underlying receptor for IgE.Fc, Or it causes the assembly of FcεRI. This receptor assembly subsequently activates signal pathways that cause granule exocytosis and release of pharmaceutical mediators such as histamine, leukotrienes, tryptase, cytokines and chemokines. This release of mediators from mast cells and basophils causes pathological signs of various allergies.

  Anti-IgE antibodies that bind to free IgE in blood and interstitial fluid and mIgE in B cells, but not to IgE bound to FcεRI on basophils and mast cells, are alleged by IgE. Developed for the treatment of disease. Treatment of the humanized anti-IgE antibody with omalizumab (trade name Xolair) exhibits multiple pharmaceutical effects in reducing type I hypersensitivity reactions in the induction of various allergies. The antibody binds IgE with high affinity at a site in the CH3 domain of Fc that overlaps with the binding site of FcεRI. Therefore, this therapy is based on the binding of the antibody to free IgE and mIgE on B lymphocytes and memory B cells, which binds to the total free IgE level in the blood and interstitial fluid. Cause a decline.

  The binding of anti-IgE to free IgE further prevents IgE from binding to FcεRI on the surface of basophils and mast cells. Since FcεRI without IgE binding is unstable and they are internalized and degraded, inhibition of the binding of free IgE to anti-IgE inhibits FcεRI on basophils and mast cells. Gradually down control. Evidence for other effects of both antibodies includes neutralization of cytokine activity, alleviation of overall inflammatory activity, and the possibility of sweeping allergies due to the accumulation of IgE-anti-IgE immune complexes.

One of the inventors of the present invention (TW Chang) found that Omalizumab is present on human mIgE for targeting B lymphocytes expressing mIgE in addition to the antigenic sites on CH3 of IgE and CεmX. It was found to bind to other antigenic sites called. CεmX is a 52 amino acid fragment located between the CH4 domain of the human membrane-bound ε chain (mε) and the C-terminal membrane anchor fragment. It has been shown that in most humans studied, mε (mε _s ) without CεmX accounts for a small percentage, while mε (mε _L ) with CεmX is predominantly expressed. The free secreted IgE ε chain and the mIgE mε _s and mε _L mRNA are all derived from alternative splicing of the εRNA transcript. The amino acid and nucleotide sequence of CεmX is unique among all protein and DNA databases. Thus, CεmX provides a specific antigenic site for targeting mIgE and mIgE expressing B cells.

Chang's research group is a recombinant protein containing a CεmX fragment, and a human myeloma-derived cell line SK0-007 cell line expressing human mIgE, and mε _L (mε _{L (CH2-CM} ); CM: cytoplasm) We have already reported the development of several CεmX-specific mouse monoclonal antibodies, including a20, that can bind to cells of the CHO cell line transfected with a gene corresponding to a fragment derived from the CH2 domain through the cytoplasmic tail of . Antibody a20 and all earlier developed antibodies were found to bind to the 8-aa peptide region RADWPGPP, residues # 45-52, at the C-terminus of 52a.a of CεmX.

Related patent documents cited
US5.091.313 2/1992 Chang
US5,254,671 10/1993 Chang
US5,260,416 11/1993 Chang
US5,274,075 12/1993 Chang
US5,292,867 3/1994 Chang
US5,342,924 8/1994 Chang
US2009 / 0010924A1 Wu

Related non-patent literature cited
Davis FM, Gossett LA, Chang TW (1991) An epitope on membrane-bound but not secreted IgE: implications in isotype-specific regulation.Bio/Technology 9: 53-56.
Peng C, Davis FM, Sun LK, Liou RS, Kim YW, Chang TW (1992) A new isoform of human membrane-bound IgE. J Immunol 148: 129-136.
Chen, HY, Liu, FT, Hou, CMH, Huang, JSW, Sharma, BB, and Chang, TW. (2002) Monoclonal antibodies against CεmX domain in human membrane-bound IgE and their potential on targeting IgE-expressing B cells. Int. Archives Allergy & Immunol. 128, 315-324.

  The present invention relates to the development and identification of antibodies specific for the CεmX domain of human mIgE and capable of binding to mIgE on human B lymphocytes. It also relates to the use of these antibodies in the treatment of allergic diseases and other diseases mediated by IgE.

In the study of anti-CεmX monoclonal antibody a20 developed by Chang's research group, a20 represents Igα (CD79a), Igβ (CD79b), CD21, CD19, CD81 and other proteins related to B cell receptor (BCR) It was found that a cell line such as a CHO cell line or an NSO cell line that was not expressed was transfected with the mεL (CH2-CM) gene and showed good binding. However, a20 is found to be weakly bound to cell lines that express Igα, Igβ, and other BCR-related proteins, such as the Ramos cell line transfected with the mεL _(CH2-CM) gene. did. Applicants speculated that the antigenic epitope on CεmX recognized by a20 was blocked by any BCR-related protein. Therefore, the a20 monoclonal antibody and its chimeric and humanized versions are not suitable for use in targeting mIgE expressing B lymphocytes and memory cells in vivo.

  Given that the peptide epitope RADWPGPP is the only epitope that induces an antibody response, all monoclonal antibodies produced by the hybridoma method using mice immunized with human CεmX-containing proteins should be specific for this peptide region. . However, given that this epitope is a dominant epitope but not the only immunogenic epitope, there is room to develop monoclonal antibodies specific for other antigens on CεmX. There may be epitopes of CεmX that do not block antibody binding by BCR-related proteins. If present, antibodies can be developed that bind to IgE on B cells and can be used to target such B cells.

  In the examples below, Applicants have successfully shown that RADWPGPP is a dominant epitope but not the only immunogenic and antigenic epitope of CεmX. In addition, Applicants have found monoclonal antibodies 4B12 and 26H2 that bind to CεmX with an antigenic epitope that is not located within the region of RADWPGPP. These monoclonal antibodies do not compete with the a20 antibody for binding to CεmX. They bind to mIgE on B cells more strongly than a20 and cause cytolysis and apoptosis of mIgE expressing cells more effectively than a20.

  This example shows that monoclonal antibodies such as 4B12 and 26H2 can bind to mIgE on human B lymphocytes and target mIgE-expressing B lymphocytes and memory B cells for downregulation of IgE synthesis. Suggests that it is suitable for use. The chimeric or humanized form of the antibody may be useful for use in patients suffering from IgE-mediated allergic diseases such as allergic asthma, allergic rhinitis and atopic dermatitis. Neutralization of IgE by anti-IgE is cooling-induced urticaria, chronic urticaria, cholinergic urticaria, chronic rhinosinusitis, systemic lipocytosis, cutaneous lipocytosis, allergic bronchopulmonary aspergillosis, recurrent idiopathic Antibodies such as 4B12 and 26H2 have been shown to treat these various diseases, as they have been shown to effectively treat angioedema and interstitial cystitis, or eosinophil-related gastrointestinal disorders Sometimes used.

  Furthermore, the example suggests the potential utility of peptides recognized by 4B12 and 26H2 in inducing an immune response against CεmX, an mIgE expressing B cell. Molecular constructs containing similar antigenic features, i.e. said peptides and their analogues having binding activity to anti-CεmX antibodies such as 4B12 and 26H2, individually or in part capable of inducing T cell help It may be used in combination with. Such constructs can induce strong immunization against mIgE-expressing B cells, thus realizing a down-regulating effect on overall IgE synthesis.

FIG. 1 shows three synthetic peptides with consecutive fragments of CεmX and the reactivity of these peptides with various anti-CεmX mAbs. Amino acid residues of the CεmX domain are shown in bold.

Figure 2 shows the binding of various anti-c [epsilon] mx mAbs to CHO or Ramos cell lines expressing MIgE.Fc _L or mIgE.Fc _s.

FIG. 3A shows that chimeric c4B12 and c26H2 induce ADCC in mIgE.Fc _L expressing Ramos cells at various E / T ratios.

FIG. 3B shows that chimeric c4B12 and c26H2 induce ADCC in a dose-dependent manner against mIgE.Fc _L- expressing Ramos cells.

FIG. 4A shows that exposure of PS by chimeric c4B12 and c26H2 in mIgE.Fc _L expressing Ramos cells is dose dependent.

FIG. 4B shows that apoptotic nuclei were observed in mIgE.Fc _L expressing Ramos cells treated with chimeric c4B12 and c26H2.

FIG. 4C shows that caspase position 3 and cleavage of PARP was observed in mIgE.Fc _L expressing Ramos cells treated with chimeric c4B12 and c26H2.

5, V _L and V _H of the parental mouse 4B12, the V _L and V _H of human germline templates KV2 and HV4 selected, and humanized 4B12 (hu4B12) (in the alignment referred to as "substitution"), Sequence alignment is shown. This hu4B12 has a binding affinity for CεmX recombinant protein and mIgE.Fc _L- expressing Ramos cells equivalent to that of chimeric 4B12 (c4B12).

Example 1: Novel anti-CεmX monoclonal antibody that binds to an antigenic site other than RADWPGPP To induce an anti-CεmX immune response, BALB / c mice were emulsified in TiterMax Gold adjuvant (Sigma-Aldrich) according to the instructions. 50 μg of n-undecyl-β-d-maltopyranoside (UDM; Anatrace) solubilized mIgE.Fc _L recombinant protein was used to immunize twice at 2-week intervals by subcutaneous administration. Applicants avoided the use of hyper-immunization protocols so that mice do not produce antibodies only against the predominant RADWPGPP epitope. 0.1 mg of UDM solubilized mIgE.Fc _L recombinant protein was administered intraperitoneally without adjuvant to give the final boost. One day prior to fusion, NSO cells were placed in fresh DMEM medium (Invitrogen) supplemented with 10% fetal bovine inactivated serum (FBS; Invitrogen) and 1% penicillin-streptomycin (100x Pen-Strep solution; Invitrogen). Reseeding was performed at a cell density of 5 × 10 ⁵ cells / ml. Three days after the last boost, spleen cells from two immunized mice were collected and washed twice with serum-free DMEM medium. 5 × 10 ⁷ NSO cells were collected and washed twice with serum-free DMEM medium. After washing, spleen cells and NSO cells are fused with 50% polyethylene glycol 1500 (PEG 1500, Roche Applied Science) pre-warmed, and gently stirred using a pipette tip for 1 minute. Stir for 1 minute, 2 ml of pre-warmed serum-free DMEM was added over 2 minutes, and finally 8 ml of pre-warmed serum-free DMEM was added over 2 minutes. After centrifugation at 200 xg for 10 minutes, the fused cells were washed with 600 ml of HAT medium (2% hypoxanthine-aminopterin-thymidine mixture (50 x HAT solution; Invitrogen), 10% BM-Condimed H1 (Roche Applied Science), The suspension was resuspended in DMEM medium supplemented with 10% inactivated FBS and 1% penicillin-streptomycin mixture, and dispensed into 30 96-well culture plates at 200 μl / well. On the third day, 100 μl of HAT medium was added to each well. On days 7 and 10, half of the medium in each well was aspirated and the same amount of HAT medium was added to refresh the medium. On day 14, the hybridoma supernatant was screened for anti-CεmX mAbs binding to UDM-solubilized mIgE.FcL or mIgE.Fcs protein by enzyme-linked immunosorbent assay (ELISA).

To screen for hybridomas secreting anti-CεmX mAbs by ELISA, purified UDM solubilized mIgE.FcL or mIgE.Fcs protein dissolved in 0.1 M NaCO ₃ (pH 9.6) at 50 ng / well in 96-well MaxiSorp plates ( Nunc) was coated overnight at 4 ° C. Coated wells were blocked with 200 μl / well of 1% BSA in PBS for 1 hour at room temperature. The plate was washed 3 times with 200 μl / well of PBS with 0.05% Tween-20. Incubation was performed at room temperature for 2 hours. The medium in all wells was aspirated and the plate was washed 6 times with 200 μl / well of PBS supplemented with 0.05% Tween-20. HRP-conjugated goat anti-mouse IgG antibody (Chemicon) diluted 1: 10000 was added to the plate (100 μl / well) and incubated for 1 hour. Subsequently, the supernatant of all wells was aspirated and the plate was washed 6 times with 200 μl / well of PBS supplemented with 0.05% Tween-20. Finally, the wells were developed with 50 μl / well of tetramethylbenzidine (TMB) substrate solution (SureBlue ™, KPL) and the reaction was stopped by adding 50 μl / well of 1N HCl. The absorbance of OD ₄₅₀ was measured on an ELISA reader. Of the more than 4000 hybridoma clones that were fusions of two cells, 17 clones were shown by ELISA to be more specific for UDM solubilized mIgE.Fc _L than mIgE.Fcs.

To investigate the specificity of anti-CεmX mAbs for CεmX, the reactivity of the multiple CεmX-specific clones to three synthetic peptides was tested. The three synthetic peptides are continuous CεmX fragments divided by a C residue located at the # 18 residue and a CHC fragment located at the # 39-41 residues. In particular, the P1 peptide contains the last 4 amino acid residues of CH4 of mε and the first 17 amino acid residues (# 1-17) of CεmX, ie GLAGSSAQSQRAPDRVL; P2 peptide contains 20 of # 19-38 of CεmX The amino acid residues, i.e. HSGQQQGLPRAAGGSVPHPR; the P3 peptide is CεmX, the terminal 11 amino acid residues (# 42-52), i.e. GAGRADWPGPP, and the first 4 amino acid residues in the continuous migis region, i.e. Contains the N-terminal extracellular region of the chain membrane anchor peptide. All peptides were synthesized at the Genomics Research Center at Academia Sinica (Taipei, Taiwan). These peptides were reconstituted to 10 mg / ml in PBS. All peptides were dissolved in 0.1 M NaCO ₃ (pH 9.6) and coated at 96 ng / well, 96 well MaxiSorp plates (Nunc) at 4 ° C. overnight. Coated wells were blocked with 200 μl / well of 1% BSA in PBS for 1 hour at room temperature. The plate was washed 3 times with 200 μl / well of PBS with 0.05% Tween-20, followed by the addition of 1OO μl of 1 μg / ml anti-CεmX mAbs antibody to the wells. Incubation was performed at room temperature for 2 hours. The medium in all wells was aspirated and the plate was washed 6 times with 200 μl / well of PBS supplemented with 0.05% Tween-20. To the plate, HRP-conjugated goat anti-mouse IgG antibody diluted 1: 10000 was added and incubated for 1 hour. Subsequently, the plate was washed 6 times with PBS 200 μl / well added with 0.05% Tween-20, and then 50 μl / well TMB substrate solution was added to each well. The reaction was stopped by adding 50 μl / well of 1N HCl. The absorbance of OD ₄₅₀ was measured on an ELISA reader. Of the many CεmX specific monoclonal antibodies prepared in Applicants' experiments, only 4B12 and 26H2 did not react with P3 peptides including RADWPGPP. 4B12 reacted with the P1 peptide and 26H2 reacted with the P2 peptide. Any other CεmX specific monoclonal antibody reacted with P3 (FIG. 1). Thus, RADWPGPP is correctly a dominant immunogenic epitope. However, it is not the only immunogenic epitope.

Example 2: Binding of 4B12 and 26H2 to mIgE on mIgE-expressing B cells Applicants further provided recombinant DNA encoding either mεL _(CH2-CM) or mεs _(CH2-CM). The performance of a number of CεmX specific monoclonal antibodies binding to transfected CHO and Ramos cell lines was tested. Transfected two CHO cell lines were were respectively produced MIgE.Fc _L or MIgE.Fc _s, but none forming a secondary receptor and full B cell receptors, such as Igα and Ig [beta]. This is because CHO cells did not express these proteins. Transfected Ramos cell line has been produced MIgE.Fc _L or MIgE.Fc _s respectively, were all formed endogenous (native) sub receptor complex. To investigate the binding of anti-CεmX mAbs to endogenous CεmX, CHO or Ramos cells expressing either mIgE.Fc _L or mIgE.Fc _s were treated with FACS buffer [PBS, 1% FBS, 0.1% sodium azide. And 2 mM EDTA (pH 8.0)] at a density of 10 ⁷ cells / ml. 10 ⁶ cells were then incubated with 100 μl of hybridoma supernatant for 30 minutes on ice, followed by washing with FACS buffer. Detection of bound antibody by analyzing FITC-labeled rabbit F (ab ') ₂ fragment-specific antibody against mouse IgG (AbD Secrotec) for 30 minutes on ice, followed by washing twice with FACS buffer did. Flow cytometry testing was performed using a FACSCanto II flow cytometer (BD Bioscience) and analysis was performed using FCSExpress software (De Novo Software). In all CεmX specific monoclonal antibodies, binding to CHO and Ramos cells expressing MIgE.Fc _s was not confirmed. All CεmX specific monoclonal antibodies were found to bind to CHO cells expressing mIgE _L. However, only 4B12 and 26H2 bound to Ramos cells expressing mIgE.Fc _L , but all other CεmX specific monoclonal antibodies failed to bind to Ramos cells expressing (FIG. 2).

Example 3 Induction of Antibody-Dependent Cytotoxicity by 4B12 and 26H2 on mIgE-expressing B Cells To investigate the ADCC activity of chimeric anti-CεmX mAbs, Applicants used peripheral blood mononuclear cells (PBMC) as effector cells. Was used to target Ramos cells expressing mIgE.Fc _L. PBMCs were purified from a healthy donor buffy coat (Taiwan Blood Service Foundation) by density gradient centrifugation using Ficoll-Paque Plus (GE Healthcare), which was purified by 90% FBS / 10% DMSO (Hybri-MaxTM). And stored frozen in Sigma-Aldrich). Prior to use, PBMCs were thawed and cultured overnight at 2 × 10 ⁶ cells / ml in MIDM medium (Invitrogen) supplemented with 10% heat-inactivated FBS and 1% penicillin-streptomycin mixture. To identify target cells co-cultured with PBMC, mIgE.Fc _L expressing Ramos cells were treated with 2.5 μM 5- (and -6) -carboxyfluorescein diacetate succinimidyl ester (0.1% BSA / PBS). CFDA, SE; Invitrogen) for 10 minutes at 37 ° C. After washing 3 times with cold RPMI medium (Invitrogen) containing 10% FBS, the cells were adjusted to 10 ⁵ cells / ml. To titrate the effector-target (E / T) ratio, 20000 labeled cells in 200 μl complete RPMI medium were exposed to 1 μg / ml antibody for 30 minutes at 37 ° C., followed by Combined with volume of PBMC and multiple E / T ratios of 50-3.125. For antibody titration, 20000 labeled cells in 200 μl of complete RPMI medium were opsonized with various concentrations of antibody (1000-0.01 ng / ml) for 30 minutes at 37 ° C., followed by E / Combined with PBMC at a T ratio of 25: 1. To measure antibody-independent cell death, labeled target cells were combined with PBMC in the absence of antibody at a given E / T ratio. At the end of the 24 hour incubation, dead cells were stained with 2.5 μg / ml 7-aminoactinomycin (7-AAD; Invitrogen) for 15 minutes on ice. Cells were analyzed on a Becton Dickinson FACSCanto II flow cytometer. Viable target cells were defined as the percentage of CFSE-positive / 7-AAD-negative on the dot plot analysis. The percentage of cells that died at a given E / T ratio was calculated according to the following formula:
100 × [(% of living target cells in antibody-independent control−% of living target cells in sample) /% of living target cells in antibody-independent control]
ADCC activity of c4B12, c26H2, and omalizumab was observed at multiple E / T ratios. When the E / T ratio is 50, c4B12, c26H2 and omalizumab caused 60% specific lysis; whereas the activity of ca20 was low and the specific lysis caused was only 20% (FIG. 3A) . Significant ADCC was observed when c4B12 and c26H2 concentrations exceeded 0.01 μg / ml. At the maximum dose of 10 μg / ml, specific lysis of target cells by c4B12 and c26H2 was 80-90%, whereas specific lysis of ca20 was 50% (FIG. 3B). The positive controls rituximab and omalizumab directed against CD20 effectively induced ADCC at multiple E / T ratios in a concentration-dependent manner. Therefore, Applicants have noted that c4B12 and c26H2 are more potent anti-CεmX mAbs than ca20 in inducing ADCC and can efficiently assemble effectors to target mIgE-expressing B cells in vivo. I can do it.

Example 4: Induction of apoptosis by chimeric anti-CεmX mAbs on membrane-bound IgE.Fc _L expressing Ramos cells To detect phosphatidylserine (PS) exposure, mIgE.Fc _L -expressing Ramos cells (5x10 ⁵ cells / ml) were incubated with chimeric anti-CεmXmAbs, omalizumab or control antibody at the indicated concentration in complete culture medium for 1 hour at 37 ° C. Subsequently, the cells were treated with a goat F (ab ′) ₂ fragment (Jackson ImmunoResearch Laboratories Inc.) specific for an Fc fragment of human IgG at a concentration of 10 μg / ml, and further incubated at 37 ° C. for 24 hours. Detection of phosphatidylserine (PS) exposure is 200 μl containing fluorescein isothiocyanate (FITC) labeled annexin V (Bio Vision) diluted to 1/200 and 2.5 μg / ml probidium iodide (PI, Sigma-Aldrich). The cells were evaluated by staining the cells in the annexin buffer for 15 minutes at room temperature in the dark. Cells were analyzed on a FACSCanto II flow cytometer. Apoptotic cells were detected as a percentage of Annexin V negative / PI positive cells on the dot plot analysis. Approximately 80% of mIgE.Fc _L- expressing Ramos cells died of apoptosis by increasing the concentration of c4B12, c26H2, or omalizumab, but in the case of ca20, up to 1 μg / ml did not (FIG. 4A).

To detect apoptotic nuclei, mIgE.Fc _L- expressing Ramos cells (5 × 10 ⁵ cell / ml) were mixed with 1 μg / ml chimeric anti-CεmX mAbs, omalizumab or control antibody in complete culture medium at 37 ° C. Incubated for hours. The cells were then treated with a goat F (ab ′) ₂ fragment specific for the Fc fragment of human IgG to a final concentration of 10 μg / ml and further incubated at 37 ° C. for 48 hours. 5xlO ⁵ cells were added to 0.5 ml of Probidium iodide (PI) / Triton solution (0.1% sodium acetate, 0.1% Triton X-100, 15 μg / ml PI, and 100 μg / ml RNase A in PBS; all Sigma- Aldrich) and incubated in the dark for 1 hour on ice. PI fluorescence was determined on a FACSCanto II flow cytometer. The content of intact nuclear DNA was recorded on a linear scale. Apoptotic nuclei containing hypodiploid DNA fluoresce in the channel below the G ₀ / G ₁ peak and are counted as a percentage of the total population. A significant increase in cell populations with low double DNA was observed in mIgE.Fc _L expressing Ramos cells treated with c4B12, c26H2 or omalizumab (FIG. 4B).

To detect caspase 3 and poly (ADP-ribose) polymerase (PARP) cleavage, mIgE.Fc _L expressing Ramos cells were cultured at 37 ° C for 1 hour in complete culture medium at a concentration of 1 μg / ml. Incubated with CεmX mAbs, omalizumab or control antibody. The cells were then treated with a goat F (ab ′) ₂ fragment specific for the Fc fragment of human IgG to a final concentration of 10 μg / ml and further incubated at 37 ° C. for 24 hours. 5xlO ⁶ cells were washed with ice cold PBS, 100 μl ice cold adjusted RIPA buffer [20 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton-X 100, 0.5% deoxycholate, 0.1% Resuspended in sodium dodecyl sulfate (SDS), 5 mM EDTA, and protease inhibitor (Sigma-Aldrich). The lysate was incubated on ice for 20 minutes. Samples were centrifuged at 16000 xg for 20 minutes at 4 ° C. The supernatant was transferred to a new 1.5 ml tube and stored at -80 ° C. The amount of protein in each clarified lysate was quantified using the Protein DC assay (Bio-Rad Laboratories) according to the instructions. Each sample was averaged by total protein amount, subjected to SDS polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred to a PVDF membrane (GE Healthcare). Rabbit polyclonal antibodies against caspase 3 and PARP were obtained from Cell Signaling Techonology and diluted 1: 500. HRP-conjugated goat anti-rabbit IgG secondary antibody (Sigma Aldrich) was diluted 1: 10000. The membrane was developed with ECL (Immobilon ™ Western; Millipore) reagent. Equivalent protein loading was assessed by Western blotting using an antibody against β-actin (Sigma-Aldrich). mIgE.Fc _L- expressing Ramos cells treated with c4B12, c26H2, and omalizumab were more prominent in caspase 3 cleavage to M _r 19 and 17 kDa fragments 24 hours after treatment than those treated with ca20 . On the other hand, cleavage of PARP, using antibodies that recognize M _x 116 kDa full PARP and M _x 89 kDa cleavage product, can be detected in MIgE.Fc _L expressing Ramos cells treated with c4B12, c26H2 and omalizumab (FIG. 4C).

Claims (7)

A pharmaceutical composition for inducing an immune response, an immunogen a therapeutically effective amount consisting GLAGGSAQSQRAPDRVL (SEQ ID NO: 2); and an adjuvant; containing, the pharmaceutical compositions. The pharmaceutical composition according to claim 1, for treating IgE-mediated diseases. The IgE-mediated diseases include cooling-induced urticaria, chronic urticaria, cholinergic urticaria, chronic rhinosinusitis, systemic lipocytosis, cutaneous lipocytosis, allergic bronchopulmonary aspergillosis, recurrent idiopathic vascularity The pharmaceutical composition according to claim 2, which is edema, interstitial cystitis, or eosinophil-related gastrointestinal disorder. The pharmaceutical composition according to claim 2, wherein the IgE-mediated disease is an allergic disease. The pharmaceutical composition according to claim 4, wherein the allergic disease is allergic asthma, allergic rhinitis, or atopic dermatitis. The pharmaceutical composition according to any one of claims 1 to 5, wherein the immune response is an antibody reaction that induces production of an antibody specific to GLAGGSAQSQRAPDRVL (SEQ ID NO: 2). 7. The pharmaceutical composition according to claim 6, wherein the antibody can bind to membrane-bound IgE on B lymphocytes and induce apoptosis of B lymphocytes.

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