anti-CmX antibodies capable of binding to mIgE on human B lymphocytesBackground
IgE plays a crucial role in mediating type I hypersensitivity reactions, which are responsible for the resulting allergic diseases, including allergic asthma, allergic rhinitis, atopic dermatitis, etc. Anaphylaxis is the response of the immune system to harmless environmental substances such as dust mites, tree and grass pollen, certain foods and pharmaceuticals, and the bite of bees and fire ants. In these reactions, binding of allergens to basophil and mast cell surface IgE causes IgE cross-linking and aggregation of the associated ige.fc receptors, which are type I ige.fc receptors or FcRI. This receptor aggregation subsequently activates signaling pathways, leading to exocytosis of the particles and the release of pharmacological mediators, such as histamine, leukotrienes, tryptase, cytokines and chemokines. The release of those mediators from mast cells and basophils leads to various pathological manifestations of the allergic reaction.
anti-IgE antibodies have been developed for the treatment of IgE-mediated allergic diseases, which bind to free IgE in blood and interstitial fluid and mlge on B cells, but not to FcRI-bound IgE on basophils and mast cells. Treatment with a humanized anti-IgE antibody, omalizumab (trade name Xolair), showed multiple pharmacological effects of weakening type I hypersensitivity in various allergic disorders. The antibody binds with high affinity to IgE at the site in the CH3 domain of Fc that overlaps with the binding site of FcRI. Thus, the therapy is based on the binding of antibodies to free IgE and mlge on B lymphoblasts and memory B cells, which results in a decrease in total free IgE levels in blood and interstitial fluid.
Binding of anti-IgE to free IgE further prevents binding of IgE to FcRI on the surface of basophils and mast cells. Depletion of free IgE with anti-IgE binding also gradually down regulates FcRI on basophils and mast cells due to instability of FcRI not occupied by IgE, and subsequent internalization and degradation. Evidence has been found for other effects of antibody therapy, including neutralization of cytokine activity, attenuation of total inflammatory activity, and possible clearance of allergens by accumulation of IgE-anti-IgE immune complexes.
One of the present inventors (t.w. chang) found that IgE is present on human mIgE in addition to the antigenic site located on CH3 to which omalizumab binds, another antigenic site called CmX, for targeting the B lymphocytes expressing mIgE. CmX is a 52 amino acid fragment located between the CH4 domain and the C-terminal membrane anchoring fragment of the human membrane bound chain (m). In most human individuals studied, it has been demonstrated that there is no m (m) of CmXS) In a very small proportion, with an m-chain (m) of CmXL) And (4) expressing superiority. Free and secretory type ImRNA of strand of gE and m of mIgESAnd mLAll of the mRNA's are derived from alternative splicing of RNA transcripts. CmX is unique throughout protein and DNA databases. CmX thus provides unique antigenic sites for targeting and expressing mIgE in B cells.
The Chang group previously reported that several CmX-specific mouse monoclonal antibodies, including a20, were developed that bind to recombinant proteins containing the CmX fragment and cells of the SKO-007 cell line, which is a human myeloma-derived cell line that expresses human mIgE, and to cells of the CHO cell line, which corresponds to mLFragment from the CH2 domain to the cytoplasmic terminus (m)L(CH2-CM)(ii) a CM: cytoplasm). Monoclonal antibody a20 and all of its earlier developed antibodies were found to bind to the 8-a.a. peptide region located C-terminal to the 52a.a.CmX domain, RADPWPGPP (SEQ ID NO: 1), residues # 45-52.
Summary of The Invention
The present invention relates to the development and identification of antibodies specific for the CmX domain of human mIgE and capable of binding to mIgE on human B lymphocytes. The invention also relates to the use of these antibodies in the treatment of allergic and other diseases mediated by IgE.
In the course of studying the anti-CmX monoclonal antibody a20 developed by Chang's research group, a20 and m were foundL(CH2-CM)Binding is preferred for cell lines transfected with genes, such as the CHO cell line or the NS0 cell line, which do not express Ig α (CD79a), Ig β (CD79B), CD21, CD19, CD81 and other B Cell Receptor (BCR) -associated proteins. However, a20 and m were foundL(CH2-CM)The cell lines transfected with the genes expressing Ig α, Ig β and other BCR-related proteins are poorly associated, such as Ramos cell lines. We hypothesized that the epitope recognized by a20 that is located on CmX might be blocked by certain BCR-related proteins. Thus, the a20 monoclonal antibody and chimeric or humanized versions thereof are not suitable for use in vivo in human patients for targeting mIgE-expressing B lymphoblasts and memory cellsThe purpose of (1).
If the peptide epitope RAWPGPP (SEQ ID NO: 1) is the only epitope for inducing an antibody response, then the monoclonal antibodies generated by the hybridoma method using mice immunized with the protein containing human CmX will be specific for that peptide region. However, if this epitope is a dominant epitope, but not the only immunogenic epitope, monoclonal antibodies specific for other epitopes on CmX can still be developed. There may be an epitope at CmX that cannot be blocked by the BCR-related protein for antibody binding. If so, antibodies that bind IgE on B cells and can be used to target these B cells can also be developed.
In the examples below, we successfully demonstrated that although RADPWPGPP (SEQ ID NO: 1) is the dominant epitope, it is not the only immunogenic and antigenic epitope at CmX. Furthermore, we found monoclonal antibodies, 4B12 and 26H2, which bind to CmX at an epitope not within the RADPWPGPP (SEQ ID NO: 1) region. Those monoclonal antibodies did not compete with the a20 antibody in binding CmX. They bind mIgE on B cells more strongly than a20 and are more effective than a20 in causing antibody-dependent cytolysis and apoptosis of mIgE-expressing cells.
The examples show that monoclonal antibodies such as 4B12 and 26H2 bind to IgE on human B lymphocytes and are suitable for targeting mIgE-expressing B lymphoblasts and memory B cells for down-regulating IgE synthesis. The antibodies in chimeric or humanized form are suitable for use in patients affected by IgE-mediated allergic diseases such as allergic asthma, allergic rhinitis and atopic dermatitis. Since neutralization of IgE by anti-IgE has been demonstrated to be effective in the treatment of cold-induced urticaria, chronic urticaria, cholinergic urticaria, chronic sinusitis, systemic mastocytosis, cutaneous mastocytosis, allergic bronchopulmonary aspergillosis, recurrent congenital angioedema, and interstitial cystitis or eosinophil-related gastrointestinal disorders, these various diseases can also be treated with antibodies such as 4B12 and 26H 2.
The examples also suggest the potential use of the peptides recognized by 4B12 and 26H2 in inducing an immune response against CmX and thus against mlge-expressing B cells. Peptides and analogues thereof having similar antigenic properties, i.e. having binding activity to the anti-CmX antibody, such as 4B12 and 26H2, may be used alone or in combination with molecular constructs further comprising a moiety capable of inducing T cell help. Such constructs are capable of inducing active immunity against mIgE expressing B cells and thus effecting down-regulation of total IgE synthesis.
One aspect of the disclosure relates to CmX-specific antibodies that bind to membrane-bound IgE on B lymphocytes, but not to RADPWPGPP peptide (SEQ ID NO: 1). In one example, such CmX-specific antibodies can be mouse monoclonal antibodies. In another example, the antibody is a chimeric antibody comprising the variable region of a mouse monoclonal antibody and the constant region of a human antibody. Alternatively, the antibody is a humanized monoclonal antibody comprising substantially the hypervariable regions of a mouse monoclonal antibody and the framework and constant regions of a human antibody. In another example, the antibody is a human antibody.
Another aspect of the disclosure relates to a fragment of the CmX-specific antibody that binds to membrane-bound IgE on B lymphocytes, but does not bind to RADPWPGPP peptide (SEQ ID NO: 1). Such antibody fragments may be Fab, F (ab') 2 or single chain Fv.
In another aspect, the disclosure provides methods of treating an IgE-mediated disease, which may be allergic asthma, allergic rhinitis, or atopic dermatitis, using any one of the antibodies described herein. In some embodiments, the IgE-mediated disease is cold-induced urticaria, chronic urticaria, cholinergic urticaria, chronic sinusitis, systemic mastocytosis, cutaneous mastocytosis, allergic bronchopulmonary aspergillosis, recurrent congenital angioedema, and interstitial cystitis or eosinophil-associated gastrointestinal disorders.
In some embodiments, the antibodies described herein bind to GLAGGSAQSQRAPDRVL (SEQ ID NO:2) or analogs having similar antigenic properties. In other embodiments, the antibodies described herein bind to HSGQQQGLPRAAGGSVPHPR (SEQ ID NO: 3) or analogs having similar antigenic properties.
Also within the scope of this disclosure are (i) a method of treatment using an immunogen comprising GLAGGSAQSQRAPDRVL (SEQ ID NO:2) or an analogue having similar antigenic properties, (ii) a method of treatment using an immunogen comprising HSGQQQGLPRAAGGSVPHPR (SEQ ID NO: 3) or an analogue having similar antigenic properties, to induce an immune response in a patient, and (iii) a method of treatment using an immunogen comprising GLAGGSAQSQRAPDRVL (SEQ ID NO:2) or an analogue having similar antigenic properties and HSGQQGLPRAAGGSVPR (SEQ ID NO: 3) or an analogue having similar antigenic properties, to induce an immune response in a patient.
Examples
Example 1: a novel anti-CmX monoclonal antibody that binds to an antigenic site other than RADPWPGPP (SEQ ID NO: 1).
To induce an anti-CmX immune response, 50. mu.g of solubilized n-undecyl-beta-d-maltopyranoside (UDM; Anatrace) mIgE. Fc was usedLBALB/c mice were immunized subcutaneously twice with the recombinant protein at two week intervals according to the manufacturer's opinion, the mIgELThe recombinant protein was emulsified in TiterMax Gold adjuvant (Sigma-Aldrich). We avoided the hyperimmunization protocol so that mice did not produce antibodies only against the dominant RADWPGPP epitope. The final boost was performed intraperitoneal without adjuvant with 0.1mg of udm solubilized mlge. fcl recombinant protein. One day prior to fusion, NS0 cells were plated at 5X 105The cell density of individual cells/ml was re-inoculated in fresh DMEM medium (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Invitrogen) and 1% penicillin-streptomycin mixture (100 XPen-Strep solution; Invitrogen). After the last 3 days of boosting, splenocytes from 2 immunized mice were collected and cultured with serum-free DMEMThe substrate was washed twice. Collecting 5X 107NS0 cells were plated and washed twice with serum-free DMEM medium. After washing, splenocytes were fused with NS0 cells by adding 1ml of pre-heated 50% polyethylene glycerol 1500(PEG1500, Roche Applied Science) over 1 minute while gently and constantly agitating the cells with a pipette tip, agitating the cells again for 1 minute, adding 2ml of pre-heated serum-free DMEM over 2 minutes, and finally adding 8ml of serum-free DMEM over 2 minutes. After centrifugation at 200 Xg for 10 minutes, 600ml of HAT medium [ DMEM medium supplemented with 2% hypoxanthine-aminopterin-thymidine mixture (50 XHAT solution; Invitrogen), 10% BM-conditioned H1(Roche Applied Science), 10% heat-inactivated FBS and 1% penicillin-streptomycin mixture (Invitrogen), was added]The fused cells were resuspended and distributed in 30 96-well culture plates at 200. mu.l/well. On day 3, 100. mu.l HAT medium was added to each well. At day 7 and day 10, the medium was refreshed by aspirating half the volume of medium per well and replacing it with HAT medium. At day 14, hybridoma supernatants were screened for mIgE. Fc solubilization with UDM by enzyme-linked immunosorbent assay (ELISA)LOr mIgESProtein-bound anti-CmX mAb.
To screen hybridomas secreting anti-CmX mabs using ELISA, purified UDM solubilized migeLOr mIgESProtein in 0.1M NaCO3(pH9.6) 96-well MaxiSorp plates (Nunc) were coated with 50 ng/well overnight at 4 ℃. The coated wells were blocked with 1% BSA in PB S at 200. mu.l/well for 1 hour at room temperature. Plates were washed three times with PBS containing 0.05% Tween-20 at 200. mu.l/well, and then 100. mu.l hybridoma supernatant was added to the wells. Incubate at room temperature for 2 hours. All wells were aspirated and the plates were washed six times with 200. mu.l/well of PBS containing 0.05% Tween-20. The plates were incubated for 1 hour (100. mu.l/well) with a 1: 10,000 dilution of HRP-conjugated goat anti-mouse IgG antibody (Chemicon). All wells were then aspirated and the plates were washed six times with 200. mu.l/well of PBS containing 0.05% Tween-20. Finally, the wells were developed by 50. mu.l/well Tetramethylbenzidine (TMB) substrate solution (SureBlueTM, KPL) and the reaction was stopped by adding 1N Cl at 50. mu.l/well. Detection of OD on microplate reader450OfAbsorbance. From > 4000 hybridoma clones screened for two fusions, 17 clones showed mIgE. Fc solubilization of UDM as determined by ELISALFc rather than mIgESThe specificity of (A).
To investigate the specificity of the anti-CmX mAb for CmX, a number of CmX specific clones were then tested for reactivity with 3 synthetic peptides representing 3 consecutive fragments of CmX separated by a C residue at residue #18 and a CHC fragment at residues # 39-41. Specifically, the P1 peptide contains the last 4 amino acid residues of CH4 and the first 17 amino acid residues of CmX of m (#1-17), i.e., GLAGGSAQSQRAPDRVL (SEQ ID NO: 2); the P2 peptide contains CmX of 20 amino acid residues #19-38, HSGQQQGLPRAAGGSVPHPR (SEQ ID NO: 3); the P3 peptide contained the terminal 11 amino acid residues (#42-52) of CmX, i.e., GAGRADWPGPP (SEQ ID NO: 4), and the first 4 amino acid residues of the contiguous migis region, i.e., the N-terminal extracellular region of the membrane-anchored peptide of the m-chain. All peptides were synthesized at the central institute of genomics research center (Taipei, Taiwan) (genomics research Cneter, academy Sinica (Taipei, Taiwan)). The peptides were rehydrated with PBS at a concentration of 10 mg/ml. All peptides were used at 0.1M NaCO3(pH9.6) 96-well MaxiSorp plates were coated with 500 ng/well overnight at 4 ℃. The coated wells were blocked with 1% BSA in PBS for 1 hour at 200. mu.l/well room temperature. The plate was washed three times with PBS containing 0.05% Tween-20 at 200. mu.l/well, and then 100. mu.l of 1. mu.g/ml anti-CmX mAb was added to the wells. Incubate at room temperature for 2 hours. All wells were aspirated and the plates were washed six times with 200. mu.l/well of PBS containing 0.05% Tween-20. The plates were incubated for 1 hour (100. mu.l/well) with a 1: 10,000 dilution of HRP-conjugated goat anti-mouse IgG antibody (Chemicon). After washing the plate six times with 200. mu.l/well of PBS containing 0.05% Tween-20, 50. mu.l/well of TMB substrate solution was added to the wells. The reaction was stopped by adding 1N HCl at 50. mu.l/well. Detection of OD on microplate reader450Absorbance of (d) in (d). Of the many CmX-specific monoclonal antibodies prepared in our experiments, only 4B12 and 26H2 did not bind to the P3 peptide containing RADWPGPP. 4B12 was reacted with P1 peptide and 26H2 was reacted with P2 peptide. All other CmX-specific monoclonal antibodies reacted with P3 (fig. 1). Therefore, there is no question ofRADPWPGPP (SEQ ID NO: 1) is a dominant immunogenic epitope. It is not the only immunogenic epitope.
Example 2: 4B12 and 26H2 bind to mIgE on mIgE expressing B cells
We further tested various mX-specific monoclonal antibodies and the antibodies using the code mL(CH2-CM)Or mS(CH2-CM)Binding capacity of CHO and Ramos cell lines transfected with recombinant DNA of (1). Two transfected CHO cell lines produced mIgE. Fc separatelyLOr mIgES，mIgE.FcLOr mIgESNeither is able to form complete B cell receptors with co-receptors (receptors) such as Ig α and Ig β, because the CHO cells do not express those proteins. Two Ramos cell lines transfected produced separately mIgE. FcLOr mIgES，mIgE.FcLOr mIgESBoth are capable of forming complexes with their natural co-receptors. To investigate the binding of the anti-CmX mAb to native CmX, mige. fc will be expressedLOr mIgESCHO or Ramos cells of (1)7Cell density of individual cells/ml was resuspended in FACS buffer [ PBS, 1% FBS, 0.1% sodium azide and 2mM EDTA (pH8.0)]In (1). Then 10 is put6Individual cells were incubated with 100 μ l hybridoma supernatant for 30 minutes on ice, followed by washing with FACS buffer. Rabbit F (ab') labelled by FITC specific for murine IgG2The fragments were incubated on (AbD secretec) ice for 30 min to detect bound antibodies, followed by two washes with FACS buffer prior to analysis. Flow cytometry experiments were performed with a facscan II flow cytometer (BD Bioscience) and analyzed with FCSExpress software (De NovoSoftware). All CmX-specific monoclonal antibodies were found to be incompatible with expression of mIgESCHO and Ramos cells. All CmX-specific monoclonal antibodies were found to be equally well expressing mIgELBinding of CHO cells (a). However, only 4B12 and 26H2 were able to express mlgeLWhile all other CmX-specific monoclonal antibodies were unable to bind to cells expressing migeLBinding of Ramos cells (fig. 2).
Example 3: 4B12 and 26H2 induced antibody-dependent cytotoxicity against mIgE expressing B cells
To investigate the ADCC activity of the chimeric anti-CmX mAb, we used Peripheral Blood Mononuclear Cells (PBMCs) as targets to express migeLThe Ramos cell of (1). PBMC were purified from a pale yellow layer (buffer coat) of a healthy donor (Taiwanblood Service Foundation) by centrifugation on a Ficoll-Paque Plus (GE Healthcare) density gradient and cryopreserved in 90% FBS/10% DMSO (hybrid-Max)TM(ii) a Sigma-Aldrich). Before use, PBMC were thawed and tested at 2X 106Individual cells/ml were cultured overnight in IMDM medium (Invitrogen) supplemented with 10% heat-inactivated FBS and 1% penicillin-streptomycin mixture. To identify target cells co-cultured with PBMC, mIgE. Fc was expressed in 0.1% BSA/PBS at 37 ℃ with 2.5. mu.M 5- (and-6) -carboxyfluorescein diacetate, succinimidyl ester (CFDA, SE; Invitrogen)LRamos cells of (3) were labeled for 10 minutes. After three washes with cold RPMI medium (Invitrogen) containing 10% FBS, the cells were adjusted to 105Individual cells/ml. For effector-target (E/T) ratio titration, 20,000 labeled cells in 200. mu.l of complete RPMI medium were coated with 1. mu.g/ml of antibody at 37 ℃ for 30 minutes and then mixed with equal volumes of PBMC at various E/T ratios from 50 to 3.125. For antibody titration, 20,000 labeled cells in 200. mu.l complete RPMI medium were conditioned with various concentrations (1000-0.01 ng/ml) of antibody at 37 ℃ for 30 minutes and then mixed with PBMC at an E/T ratio of 25: 1. To determine antibody-independent killing, labeled target cells were also incubated with PBMCs at a given E/T ratio in the absence of antibody. At the end of the 24 hour incubation, the dead cells were stained with 2.5. mu.g/ml 7-amino-actinomycin (7-AAD; Invitrogen) on ice for 15 minutes. 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 cells on dot blot analysis. The percentage of killer cells at a given E/T ratio was calculated according to the following formula: 100 × [ (% of live target cells in antibody-independent control-% of live target cells in sample%)/% of live target cells in antibody-independent control%]. Observed at multiple E/T ratiosADCC activity of c4B12, c26H2 and omalizumab. At an E/T ratio of 50, c4B12, c26H2 and omalizumab gave up to 60% specific lysis; in contrast, ca20 was less active, giving only 10-20% specific cleavage (FIG. 3A). Furthermore, significant ADCC was observed when the concentrations of c4B12 and c26H2 were above 0.01 μ g/ml. At a maximum dose of 10 μ g/ml, the specific lysis of target cells by c4B12 and c26H2 ranged from 80% to 90%, whereas ca20 gave up to 50% specific lysis (fig. 3B). Positive controls for CD20 rituximab and omalizumab effectively induced ADCC in a dose-responsive manner at multiple E/T ratios. Thus, we hypothesized that c4B12 and c26H2 are more efficient anti-CmX mabs relative to ca20 in mediating ADCC, and that c4B12 and c26H2 are able to efficiently recruit effector cells to target mIgE-expressing B cells in vivo.
Example 4: fc of chimeric anti-CmX mAb induced expression of membrane bound IgELApoptosis of Ramos cells of (1)
To detect Phosphatidylserine (PS) exposure, mlge. fc will be expressed in complete mediumLRamos cells (5X 10)5Individual cells/ml) were incubated with the indicated concentrations of chimeric anti-CmX mAb, omalizumab or control antibody for 1 hour at 37 ℃. Goat F (ab') specific for human IgG Fc fragment was then used2The fragment (Jackson ImmunoResearch Laboratories Inc.) treated the cells at a concentration of 10. mu.g/ml and incubated at 37 ℃ for a further 24 hours. By incubating 200. mu.l Annexin buffer (Annexin buffer) [10mM HEPES/NaOH (pH7.4), 140mM NaCl, 5mM CaCl at room temperature in the dark2]Cells were stained for 15 minutes and assayed for Phosphatidylserine (PS) exposure, the annexin buffer containing Fluorescein Isothiocyanate (FITC) -labeled annexin v (biovision) diluted at 1/200 and 2.5 μ g/ml propidium iodide (PI, Sigma-Aldrich). Cells were analyzed on a facscan II flow cytometer. Apoptotic cells were defined as the percentage of annexin V-positive/PI-negative cells on dot blot analysis. Fc, about 80% expressed as c4B12, c26H2, or omalizumab concentration increasedLThe Ramos cells of (A) die by apoptosis and at the same timeThere was maximum induction at 1. mu.g/ml, but ca20 was not (FIG. 4A).
To detect apoptotic nuclei, mIgE. Fc will be expressedLRamos cells (5X 10)5Individual cells/ml) were incubated with chimeric anti-CmX mAb, omalizumab or control antibody at a concentration of 1 μ g/ml in complete medium at 37 ℃ for 1 hour. Goat F (ab') specific for human IgG Fc fragment was then used2The fragments were treated with cells at a final concentration of 10. mu.g/ml and incubated for a further 48 hours at 37 ℃. Will be 5X 105Cells were incubated for 1 hour on ice in the dark in 0.5ml of Propidium Iodide (PI)/Triton solution (PBS containing 0.1% sodium citrate, 0.1% Triton X-100, 15. mu.g/ml PI and 100. mu.g/ml RNaseA; all from Sigma-Aldrich). PI fluorescence was measured on a facscan II flow cytometer. The DNA content in intact nuclei was recorded on a linear scale. Apoptotic nuclei containing hypodiploid DNA emitting fluorescence below the G0/G1 peak in the channel were calculated as a percentage of the total population. Expression of mIgE. Fc in c4B12, c26H2 or omalizumab treatmentsLIn Ramos cells of (3), a significant increase in the population of cells with hypodiploid DNA was observed (fig. 4B).
To detect caspase 3(caspase3) and poly (ADP-ribose) polymerase (PARP) cleavage, mIgE. Fc will be expressed in complete mediumLRamos cells (5X 10)5Individual cells/ml) were incubated with chimeric anti-CmX mAb, omalizumab or control antibody at a concentration of 1 μ g/ml for 1 hour at 37 ℃. Goat F (ab') specific for human IgG Fc fragment was then used2The fragments were treated with cells at a final concentration of 10. mu.g/ml and incubated at 37 ℃ for a further 24 hours. Wash 5X 10 with ice-cold PBS6Cells, resuspended in 100. mu.l of ice-cold modified RIPA lysis buffer [20mM Tris (pH7.4), 150mM NaCl, 1% Triton-X100, 0.5% deoxycholic acid, 0.1% Sodium Dodecyl Sulfate (SDS), 5mM EDTA and protease inhibitor (Sigma-Aldrich)]In (1). The lysate was incubated on ice for 20 minutes. The samples were centrifuged at 16000 Xg for 20 min at 4 ℃. The supernatant was transferred to a new 1.5ml tube and stored at-80 ℃. Measured with DC protein (Bio-Rad Laboratories) according toThe amount of protein in each clarified lysate was quantified as recommended by the manufacturer. The total protein content of each sample was normalized and subjected to SDS polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred onto a PVDF membrane (GE Healthcare). Rabbit polyclonal antibodies to caspase3 and PARP were obtained from CellSignaling Techology and used at a 1: 500 dilution. HRP-conjugated goat anti-rabbit IgG secondary antibody (Sigma-Aldrich) was used at a dilution of 1: 10,000. The membrane was developed with ECL reagent (Immobilon TM Westem; Millipore). The loading of equal amounts of protein was verified by probing the blots with an antibody to beta actin (Sigma-Aldrich). Fc expression upon treatment with c4B12, c26H2 and omalizumab instead of ca20L24 hours later on in Ramos cells caspase3 clearly cleaves into Mr 19-and 17-kDa fragments. Furthermore, expression of mIgE. Fc in c4B12-, c26H 2-and omalizumab treatmentLIn Ramos cells of (2), using recognition MrCleavage of PARP was detectable by antibodies to intact PARP of 116kDa and cleavage products of Mr89kDa (FIG. 4C).
Brief description of the drawings
Figure 1 shows 3 synthetic peptides representing a contiguous fragment of CmX, and the reaction of various anti-CmX mabs with these peptides. The amino acid residues of the CmX domain are shown in bold, CH 4-migis: SEQ ID NO: 11; pl: SEQ ID NO: 12; p2: SEQ ID NO: 3; p4: SEQ ID NO: 13.
FIG. 2 shows various anti-CmX mAbs and expression of mIgELOr mIgESBinding of the CHO or Ramos cell line of (1).
FIG. 3A shows that the chimeric c4B12 and c26H2 were induced at multiple E/T ratios for expression of mIgE. FcLThe Ramos cell induced ADCC. Fig. 3B shows that chimerism c4B12 and c26H2 induce fc against expression mlge. in a dose-responsive mannerLADCC of Ramos cells.
Fig. 4A shows that the chimeras c4B12 and c26H2 are expressing mlgeLThe induced PS exposure in Ramos cells of (a) is dose dependent. FIG. 4B shows mIgE. Fc expression treated in chimerism c4B12 and c26H2LObserved in Ramos cellsAnd (4) cell nucleus. FIG. 4C shows mIgE. Fc expression treated in chimerism C4B12 and C26H2LCleavage of caspase3 and PARP was observed in Ramos cells of (a).
Figure 5 shows an amino acid sequence alignment of the selected human germline templates KV2 and HV4 for VL and VH, respectively, of parental mouse 4B12 and humanized 4B12(hu4B12), humanized 4B12(hu4B12) being labeled "Replace" in the alignment. This hu4B12 has the same binding affinity as chimeric 4B12(c4B12) for CmX recombinant protein and Ramos cells expressing mlge. fcl, 4B12 light chain: SEQ ID NO: 5; 4B12 heavy chain: SEQ ID NO: 8; KV2 light chain: SEQ ID NO: 6; HV4 heavy chain: SEQ ID NO: 9; hu4B12 (substituted) light chain: SEQ ID NO: 7; hu4B12 (substituted) heavy chain: SEQ ID NO: 10.
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