A chimeric mouse-human antibody that retains specificity for HIV gp120 and mediates the lysis of HIV-infected cells

Murine mAb BAT123, which was made against the envelope glycoprotein gp120 of HTLV-IIIB strain of HIV type 1 (HIV-1), is capable of neutralizing HTLV-IIIB in vitro. It also inhibits the fusion between uninfected CD4+ cells and HIV-1-infected cells to form syncytia. As a step to explore the potential utility of the anti-HIV antibody in vivo, we have constructed a mouse-human chimeric antibody by rDNA techniques. The chimeric antibody, which bears the variable domains of mouse antibody BAT123 and constant domains Cr1 and C kappa of human Ig retains the Ag specificity of BAT123 as determined by its reactivity with HIV-1-infected H9 cells, gp120 in Western blot analysis, and the oligopeptide recognized by BAT123. The antiviral activities of the chimeric antibody in neutralizing HIV-1 infection as well as inhibiting the syncytia formation are also found identical to those of the parent murine antibody. Moreover, in the presence of human blood mononuclear cells, the chimeric antibody but not BAT123 (mouse IgG1) induces antibody-dependent cellular cytotoxicity. The findings point to the potential usefulness of the chimeric antibody in treating patients infected with HIV-1.     

Monoclonal anti-idiotypic antibody mimicking the principal neutralization site in HIV-1 GP120 induces HIV-1 neutralizing antibodies in rabbits

A murine mAb BAT123 (Ab1) directing to the principal neutralization site of human T cell leukemia virus (HTLV)-IIIB gp120 (amino acid residue 308-322) was used to generate syngeneic anti-Id mAb (Ab2). Among the Ab2, a mAb AB19-4 was characterized by both serologic and biologic methods to be paratope-specific (Ab2 beta), bearing the internal image of the neutralization site. AB19-4 was found to bind specifically to BAT123 and also to its mouse-human chimeric form in ELISA. The binding of AB19-4 to BAT123 was specifically inhibited by HTLV-IIIB gp120 and the synthetic epitope peptides of HTLV-IIIB and HTLV-IIIMN defined by BAT123. AB19-4 also inhibited the binding of BAT123 to HTLV-IIIB-infected H9 cells in flow cytometric studies. Polyclonal goat and sheep antisera against HTLV-IIIB gp120 reacted specifically with AB19-4, suggesting that AB19-4 may recognize cross-species idiotopes. Rabbits immunized with purified AB19-4 generated anti-anti-Id antibodies (Ab3) that reacted specifically with HTLV-IIIB gp120 and the BAT123-binding epitope peptides of HTLV-IIIB and HTLV-IIIMN. The Ab3 bound to H9 cells infected by HTLV-IIIB or HTLV-IIIMN and inhibited the infection of CEM cells by HTLV-IIIB or HTLV-IIIMN, whereas BAT123 also bound H9 cells infected by HTLV-IIIB or HTLV-IIIMN but neutralized only HTLV-IIIB. Our data suggest that AB19-4 mimics the neutralization site on HIV-1 gp120 defined by BAT123. The induction of immunity to HIV using internal-image Ab2 to HIV-neutralizing antibodies may provide a viable approach for developing effective vaccines for AIDS.     

Construction, expression, and biologic activity of murine/human chimeric antibodies with specificity for the human alpha/beta T cell receptor.

Murine/human chimeric antibodies with specificity for the human TCR-alpha/beta have been produced by genetic engineering. The L and H chain V region exons encoding the murine mAb BMA 031 were isolated and inserted into mammalian expression vectors containing the human kappa and gamma 1 or gamma 4 C region exons. The chimeric genes were transfected into murine Sp2/O hybridoma cells by electroporation and transfectomas secreting chimeric antibody were isolated. Secretion levels ranged from 1 to 7 pg/cell/24 h. The chimeric antibodies bound specifically to T cells and competed effectively with the parental murine mAb for binding to these sites. The ability to promote antibody-dependent cell-mediated cytolysis was significantly enhanced in the chimeric antibodies as compared with murine BMA 031. C-dependent cytolysis, however, was not detectable with any of the antibodies. Chimeric BMA 031 is a clinically relevant, genetically engineered antibody with potential uses in transplantation, graft-vs-host disease, autoimmune diseases and other T cell-related disorders.     

Chimeric and humanized antibodies with specificity for the CD33 antigen.

L and H chain cDNAs of M195, a murine mAb that binds to the CD33 Ag on normal and leukemic myeloid cells, were cloned. The cDNAs were used in the construction of mouse/human IgG1 and IgG3 chimeric antibodies. In addition, humanized antibodies were constructed which combined the complementarity-determining regions of the M195 antibody with human framework and constant regions. The human framework was chosen to maximize homology with the M195 V domain sequence. Moreover, a computer model of M195 was used to identify several framework amino acids that are likely to interact with the complementarity-determining regions, and these residues were also retained in the humanized antibodies. Unexpectedly, the humanized IgG1 and IgG3 M195 antibodies, which have reshaped V regions, have higher apparent binding affinity for the CD33 Ag than the chimeric or mouse antibodies.     

A novel strategy for producing chimeric bispecific antibodies by gene transfection

We have used genetic manipulation to produce chimeric bispecific antibodies. Plasmids containing variable regions of immunoglobulin from a murine hybridoma secreting anti-hepatitis B surface antigen were joined to human constant regions. These chimeric plasmids were introduced into transfectomas, secreting chimeric antibodies to iodo-hydroxy-nitrophenyl, by electroporation. Transfectomas secreting bispecific antibodies were identified. This approach has advantages over the fusion of hybridomas or chemical linking of two antibody molecules and will enable the use of bispecific antibodies in vivo.     

Construction, expression and characterization of humanized antibodies directed against the human alpha/beta T cell receptor.

Completely humanized antibodies with specificity for the human alpha/beta TCR have been produced by genetic engineering. The L and H chain V region exons encoding the murine mAb BMA 031 CD regions and human EU framework regions were synthesized and replaced into previously isolated genomic fragments. These fragments were inserted into mammalian expression vectors containing the human kappa and gamma 1 C region exons. Two variants were constructed each containing selected BMA 031 amino acids within the human frameworks. The humanized genes were transfected into Sp2/0 hybridoma cells by electroporation and transfectomas secreting humanized antibody were isolated. Levels of antibody expression up to 7 pg/cell/24 h were obtained. The humanized antibody, BMA 031-EUCIV2, competed poorly with murine BMA 031 for binding to T cells. BMA 031-EUCIV3, however, bound specifically to T cells and competed effectively with both the murine BMA 031 antibody and a previously constructed chimeric BMA 031 antibody for binding to these cells. The relative affinity of BMA 031-EUCIV3 was about 2.5 times lower than BMA 031. The ability to promote antibody dependent cell-mediated cytolysis was significantly enhanced with the engineered antibodies as compared to murine BMA 031. Humanized BMA 031 is a clinically relevant, genetically engineered antibody with potential uses in transplantation, graft vs host disease, and autoimmunity.     

Comparison of rat and rat-mouse chimeric anti-murine CD4 antibodies in vitro. Chimeric antibodies lyse low-density CD4+ cells

GK1.5, a rat anti-mouse CD4 mAb, is effective in the treatment of several autoimmune syndromes, induces tolerance to co-administered Ag, and prolongs allograft survival. We have constructed a family of molecules with GK1.5 V regions and mouse gamma 1, gamma 2a, gamma 2b, or gamma 3 constant regions to investigate the mechanisms underlying the effectiveness of GK1.5. The rat-mouse chimeric antibodies are specific for murine CD4 and have identical binding curves as rat GK1.5 on CD4+ T cells. The chimeric GK1.5 gamma 2a, GK1.5 gamma 2b, and GK1.5 gamma 3 antibodies are more efficient than rat GK1.5 at C-mediated cytotoxicity. This is attributed to the enhanced capacity of the chimeric antibodies, compared to rat GK1.5, to lyse CD4+ cells with a low cell surface Ag density. This observation may have important implications for therapy.     

Cloning and high level expression of a chimeric antibody with specificity for human carcinoembryonic antigen

A mouse/human chimeric antibody has been constructed by using variable light and variable heavy regions from a murine hybridoma specific for human carcinoembryonic antigen (CEA) (CEM231.6.7). These V regions were combined with kappa and gamma-1 constant region genes cloned from human lymphocytes. The chimeric constructs were sequentially electroporated into murine non-Ig-producing myeloma (P3.653) and hybridoma (SP2/0) cell. Significant differences were seen in expression levels between the two cell types. High levels of expression (24 to 32 micrograms/ml/10(6) cells) were seen with several of the anti-CEA SP2/0 transfectomas but not with the P3.653 cells. The SP2/0 transfectoma lines were adapted to serum-free, chemically defined media and grown in large scale fermentation cultures where they continued to secrete high levels of antibody. The chimeric antibodies remain reactive against human CEA with affinity constants comparable to that of the parental hybridoma antibody. High level expression will make practical the production of chimeric antibodies for in vivo therapeutic and diagnostic purposes.     

Cytotoxic T-cells specific for natural IgE peptides downregulate IgE production

Immunoglobulin E (IgE) plays a central role in IgE-mediated immediate type hypersensitivity. Since production of IgE depends on Th2, efforts to block IgE production and control allergic reactions include tolerization of Th2 or deviating development of Th2. We hypothesized that cytotoxic T lymphocytes targeting natural IgE peptides/MHC I complexes can eliminate IgE-producing cells and inhibit centrally IgE production. CTL to self-IgE peptides were elicited in mice immunized with nonameric p109-117, p113-121, and p103-141 (CHepsilon2 domain), which encompass both peptides with an OVA helper peptide (OVAp restricted for H-2d/b) in liposomes and presented by dendritic cells (DC). CTL from BALB/c lysed IgE peptide-pulsed P815 target as well as IgE-producing 26.82 hybridomas (H-2d). Natural tolerance to self-IgE peptides was tested in IgE sufficient (IgE +/+) as well as IgE-deficient (IgE -/-) 129/SvEv mice (H-2b). Comparable magnitude of CTL responses was observed in both strains immunized with p109-117 or p103-141 concomitantly with CD4 T-cell costimulation. CTL from 129/SvEv lysed not only IgE peptide-pulsed EL-4 but also IgE-producing B4 hybridomas (H-2b). This observation strongly suggests a correspondence of epitope of immunogenic peptide to that of physiologically processed IgE peptides presented on IgE-producing cells. Moreover, CTL were generated in 129/SvEv, immunized with the recombinant antigenized antibody in liposomes encompassing p107-123, p109-117, and p113-121 expressed in CDR3 of VH62/human gamma1. Polyclonal IgE production was inhibited by coincubation with MHC I-restricted CTL in vitro. Furthermore, antigen-specific IgE responses were inhibited in mice, immunized with p109-117 and p103-141 while IgG responses were not suppressed. Since IgE peptide sequences of CHepsilon2 are ubiquitous to all murine IgE heavy chain, peptides made as such can serve as a universal IgE vaccine to prevent allergy for a myriad of allergens in rodents. This observation suggests that similar human IgE peptides should be identified and employed to downregulate human IgE production.     

Bacterial expression and characterization of a novel human anti-IgE scFv fragment

Antibodies highly specific to human immunoglobulin (Ig) E are capable of selectively blocking the IgE interaction or eliminating IgE-producing cells, thus providing valuable agents for diagnostics and treatment of various allergic illness. An example is omalizumab, a humanized monoclonal anti-IgE antibody that is approved for the treatment of patients with moderate-to-severe allergic diseases in the US, European Union and other countries. Here, we describe the generation and characterization of a novel human anti-IgE as a single-chain antibody fragment (scFv). The bacterially-synthesized scFv showed high affinity (86 nM) and specificity to the Fc region of human IgE. To our knowledge, this is the first report of the production of a human anti-IgE scFv in E. coli. Its further development as a potential candidate for medical applications is discussed.Key words: anti-IgE, E. coli expression, scFv, antibody engineering, human antibodies, allergy diseases, antibody therapeutics     

Relationships between epitopes on IgE recognized by defined monoclonal antibodies and by the FC epsilon receptor on basophils

The present study investigated whether the sites on the FC region of the IgE molecule, recognized by different anti-IgE monoclonal antibodies (mAb), are identical to those recognized by the Fc receptor (Fc epsilon R). The anti-IgE mAb recognize different clusters of epitopes on the Fc region of IgE and could interfere to different degrees with the binding of IgE to mast cells and basophils, but still recognized cell-bound IgE. Analysis of the stoichiometry and affinity binding of 125I anti-IgE mAb Fab' to free IgE have revealed that anti-IgE mAb of one group (51.3) recognized three repetitive determinants on the IgE Fc portion, and another group (95.3) recognized only one determinant. When these stoichiometric studies were performed with cell-bound IgE, it was found that only one of the sites recognized by 51.3 mAb was involved in the Fc epsilon R binding site. On the other hand, the site recognized by 95.3 mAb was not the Fc epsilon R binding site. Such findings establish mAb 51.3 as a useful tool for isolating the IgE peptides involved in the binding site to the receptor.     

Anti-human IgG causes basophil histamine release by acting on IgG-IgE complexes bound to IgE receptors.

We have reexamined the ability of anti-human IgG antibodies to induce histamine release from human basophils. A panel of purified murine mAbs with International Union of Immunological Societies-documented specificity for each of the four subclasses of human IgG was used. Of the 24 allergic subjects studied, the basophils of 75% (18/24) released greater than 10% histamine to one or more anti-IgG1-4 mAb, whereas none of the 13 nonatopic donor's basophils released histamine after stimulation with optimal amounts of anti-IgG mAb. The basophils of 85% (11/13) of the nonatopic donors did respond to anti-IgE challenge, as did 92% (22/24) of the atopic donor cells. Histamine release was induced most frequently by anti-IgG3, and 10/18 anti-IgG responder cells released histamine with mAb specific for two or more different subclass specificities. The rank order for induction of histamine release was anti-IgG3 greater than anti-IgG2 greater than IgG1 greater than anti-IgG4. As in our previous study using polyclonal anti-IgG, 100- to 300-micrograms/ml quantities of the anti-IgG mAb were required for maximal histamine release, about 1000-fold higher than those for comparable release with anti-human IgE. Specificity studies using both immunoassays and inhibition studies with IgE myeloma protein indicated that anti-IgG induced histamine release was not caused by cross-reactivity with IgE. Ig receptors were opened by lactic acid treatment so that the cells could be passively sensitized. Neither IgE myeloma nor IgG myeloma (up to 15 mg/ml) proteins could restore the response to anti-IgG mAb. However, sera from individuals with leukocytes that released histamine upon challenge with anti-IgG mAb could passively sensitize acid-treated leukocytes from both anti-IgG responder and nonresponder donors for an anti-IgG response. The only anti-IgG mAb that induced release from these passively sensitized cells were those to which the serum donor was responsive. Sera from non-IgG responders could not restore an anti-IgG response. These data led to the hypothesis that the IgG specific mAb were binding to IgG-IgE complexes that were attached to the basophil through IgE bound to the IgE receptor. This was shown to be correct because passive sensitization to anti-IgG could be blocked by previous exposure of the basophils to IgE. We conclude that anti-IgG-induced release occurs as a result of binding to IgG anti-IgE antibodies and cross-linking of the IgE receptors on basophils.     

Structural and functional analysis of monomorphic determinants recognized by monoclonal antibodies reacting with the HLA class I alpha 3 domain.

To identify mAb reacting with the HLA class I alpha 3 domain, 14 mAb recognizing monomorphic determinants expressed on HLA-A, B, and C Ag or restricted to HLA-B Ag were screened in indirect immunofluorescence with mouse L cells expressing HLA-B7/H-2Kb chimeric Ag. mAb CR1S63, CR10-215, CR11-115, and W6/32 were found to react with the HLA class I alpha 3 domain in addition to the alpha 2 domain. mAb Q1/28 and TP25.99 were found to react only with the HLA class I alpha 3 domain. The determinants recognized by the six mAb were mapped on the HLA class I alpha 3 domain by indirect immunofluorescence staining of L cells expressing H-2Kb Ag containing different segments of the HLA-B7 alpha 3 domain chimerized with the H-2Kb alpha 3 domain. mAb TP25.99 reacts with chimeric Ag containing the HLA-B7 184 to 199 stretch, mAb CR10-215 and CR11-115 react with chimeric Ag containing the HLA-B7 184 to 246 stretch, mAb CR1S63 and Q1/28 react with chimeric Ag containing the HLA-B7 184 to 256 stretch, and mAb W6/32 reacts with chimeric Ag containing the whole HLA-B7 alpha 3 domain. Functional analysis using human CD8 alpha-bearing mouse H-2Kb-specific T cell hybridoma cells (HTB-Leu2) showed that only mAb TP25.99 inhibited IL-2 production by HTB-Leu2 cells stimulated with L cells expressing KbKbB7 Ag. This inhibition may occur because of the spatial proximity of the determinant defined by mAb TP25.99 to the CD8 alpha binding loop and/or because of change(s) in the conformation of the CD8 alpha binding loop induced by the binding of mAb TP25.99 to the HLA class I molecule. Furthermore, mAb TP25.99 inhibited the cytotoxicity of CD8-dependent and CD8-independent CTL clones. These results indicate that mAb TP25.99 has unique specificity and functional characteristics. Therefore it represents a valuable probe to characterize the role of the HLA class I alpha 3 domain in immunologic phenomena.     

Molecular basis for nonanaphylactogenicity of a monoclonal anti-IgE antibody

IgE Abs mediate allergic responses by binding to specific high affinity receptors (FcepsilonRI) on mast cells and basophils. Therefore, the IgE/FcepsilonRI interaction is a target for clinical intervention in allergic disease. An anti-IgE mAb, termed BSW17, is nonanaphylactogenic, although recognizing IgE bound to FcepsilonRI, and interferes with binding of IgE to FcepsilonRI. Thus, BSW17 represents a candidate Ab for treatment of IgE-mediated disorders. By panning BSW17 against random peptide libraries displayed on phages, we defined mimotopes that mimic the conformational epitope recognized on human IgE. Two types of mimotopes, one within the Cepsilon3 and one within the Cepsilon4 domain, were identified, indicating that this mAb may recognize either a large conformational epitope or eventually two distinct epitopes on IgE. On the basis of alignments of the two mimotopes with the human IgE sequence, we postulate that binding of BSW17 to the Cepsilon3 region predominantly blocks binding of IgE to FcepsilonRI, leading to neutralization of IgE. Moreover, binding of BSW17 to the Cepsilon4 region may explain how BSW17 recognizes FcepsilonRI-bound IgE, and binding to this region may also interfere with degranulation of IgE sensitized cells (basophils and mast cells). As a practical application of these findings, mimotope peptides coupled to a carrier protein may be used for the development of a peptide-based anti-allergy vaccine by induction of anti-IgE Abs similar to the current approach of using humanized nonanaphylactogenic anti-IgE Abs as a passive vaccine.     

A murine–human chimeric IgG antibody against vascular endothelial growth factor receptor 2 inhibits angiogenesis in vitro

Vascular endothelial growth factor receptor 2 (VEGFR2) has been reported to play an important role in angiogenesis and tumorigenesis. A murine anti-VEGFR2 mAb (A8H1) has been established in a previous study. To reduce the incompatibility of the murine mAb for human use, the chimeric anti-VEGFR2-IgG was developed by genetic recombination of the variable regions of the A8H1 antibody and the constant regions of human IgG, and was expressed in Sp2/0 cells transfected with the two recombinant vectors containing the heavy chain and the light chain regions. After screening, clone 2F12 was selected and was found to stably secrete the murine–human chimeric anti-VEGFR2-IgG (coded 2F12). This chimeric IgG maintained the specificity and the affinity of the parental murine antibody against VEGFR2, and effectively identified VEGFR2 expressed on the surface of HUVECs and BEL-7402 cells. Furthermore, the 2F12 antibody demonstrated inhibition of angiogenesis in vitro, such as proliferation, migration, invasion and tube formation of HUVECs. This murine–human chimeric IgG may be considered for further development as an anti-angiogenesis and anti-tumor agent.     

Therapy with monoclonal antibodies. An in vivo model for the assessment of therapeutic potential.

A set of rat-human and rat-rat chimeric mAb has been created, all possessing V regions identical in their specificity for the mouse CD8 Ag. In vitro all antibodies were able to block cell-mediated lysis but varied greatly in their capacity to utilize rabbit complement. We examined the ability of these chimeric antibodies to deplete in vivo and established a clear hierarchy. Of the human IgG subclasses, only IgG1, 2, and 3 could fix complement in vitro, yet all (IgG1-4) were remarkably potent at depleting CD8+ PBL in vivo. In contrast, human IgA2 and IgE were ineffective at clearing CD8+ PBL. The vector system used to create these antibodies together with the small doses of antibodies required to deplete in vivo make this a simple and rapid system for testing the effects of different antibody isotypes and mutants. We have shown that a mutant of human IgG1, which is incapable of fixing complement, depletes perfectly well in vivo, whereas an aglycosyl IgG1 mutant is rendered inactive. Our model provides a unique opportunity to study effector functions and motifs that are used by mAb in vivo and will help in the design of improved antibodies for human therapy.     

Long term maintenance of IgE-mediated memory in mast cells in the absence of detectable serum IgE

Mast cells and basophils involved in allergic responses do not have clonotypic Ag receptors. However, they can acquire Ag specificity through binding of Ag-specific IgE to FcepsilonRI expressed on their surface. Previous studies demonstrated that IgE binding induced the stabilization and accumulation of FcepsilonRI on the cell surface and resulted in up-regulation of FcepsilonRI. In this study we have further analyzed the maintenance of IgE-mediated memory in mast cells and basophils in vivo by comparing kinetics of serum IgE levels, FcepsilonRI expression, and ability to induce systemic anaphylaxis. A single i.v. injection of trinitrophenyl-specific IgE induced 8-fold up-regulation of FcepsilonRI expression on peritoneal mast cells in B cell-deficient (micro m(-/-)) mice. Serum IgE levels became undetectable by day 6, but the treatment of mice with anti-IgE mAb induced a significant drop in body temperature on days 14, 28, and 42. The administration of trinitrophenyl -BSA, but not BSA, in place of anti-IgE mAb gave similar results, indicating the Ag specificity of the allergic response. This long term maintenance of Ag-specific reactivity in the allergic response was also observed in normal mice passively sensitized with IgE even though the duration was shorter than that in B cell-deficient mice. The appearance of IgE with a different specificity did not interfere with the maintenance of IgE-mediated memory of mast cells and basophils. These results suggest that IgE-mediated stabilization and up-regulation of FcepsilonRI enables mast cells and basophils not only to acquire Ag specificity, but also to maintain memory in vivo for lengthy periods of time.     

Species specificity in the interaction of CD8 with the alpha 3 domain of MHC class I molecules.

The alpha 1 and alpha 2 domains of the class I MHC molecule constitute the putative binding site for processed peptides and the TCR, although the alpha 3 domain has been implicated as a binding site for the CD8 molecule. Species specificity in the binding of CD8 to the alpha 3 domain has been suggested as an explanation for the low xenogeneic T cell response to class I molecules, but results on this point have been conflicting and controversial. We have addressed this issue using CTL lines from HLA-A2.1 transgenic mice that specifically recognize and lyse A2.1-expressing cells infected with influenza A/PR/8 or pulsed with influenza matrix peptide M1(57-68). Species specificity was examined using transfectants that expressed hybrid molecules containing the alpha 1 and alpha 2 domains from HLA-A2.1 and the alpha 3 domain from a murine class I molecule. Lower levels of M1(57-68) peptide were required to sensitize L cell transfectants expressing a chimera that contained an H-2Dd alpha 3 domain than targets expressing the intact A2.1 molecule. However, at high doses of peptide, lysis of these two targets was similar. However, no reproducible difference in sensitization was observed using EL4 or Jurkat transfectants expressing A2.1 or A2.1 chimeric molecules that contained an H-2Kb alpha 3 domain. In all cases, however, lysis of peptide-pulsed A2.1 expressing targets was more sensitive to inhibition with anti-CD8 mAb than lysis of cells expressing these chimeric molecules. Thus, under suboptimal conditions such as low Ag density or in the presence of anti-CD8 mAb, these CTL preferentially recognize class I molecules with a murine alpha 3 domain. This suggests that there is some species specificity in the interaction of CD8 with the alpha 3 domain of the class I molecule. However, CTL recognition was inhibited by point mutations in the alpha 3 domain of HLA-A2.1 that have been shown to inhibit binding of human CD8 and recognition by human CTL, suggesting that murine CD8 interacts to some degree with human alpha 3 domains, and that similar alpha 3 domain residues may be important for murine and human CD8 binding. The relevance of these results to an understanding of low xenogeneic responses is discussed.     

Stability of murine, chimeric and humanized antibodies against pre-S2 surface antigen of hepatitis B virus

We have constructed a humanized antibody with specificity for the pre-S2 surface antigen of hepatitis B virus (HBV) by grafting the complementarity determining regions (CDRs) of parental murine monoclonal antibody (mAb) into human anti-Sm antibody framework regions. The humanized antibody has a substitution at position 94 in a framework region of the heavy chain variable region, and exhibits the same antigen binding affinity as the parental murine monoclonal and chimeric antibodies. In order to assess the stability of these antibodies, thermal inactivation of the parental, chimeric and humanized antibodies was analyzed. Fifty percent inactivation of the chimeric and humanized antibodies was observed at 63.7 degrees C and 68.7 degrees C, respectively, compared to 55.0 degrees C for murine antibody. The humanized antibody also exhibited increased stability against denaturant. Guanidine-induced unfolding monitored by the changes in fluorescence intensity at 360 nm showed that midpoints of the transition of the chimeric and humanized antibodies were 2.47 M and 2.56 M, respectively, whereas that of the murine antibody was 1.36 M.     

Bacterial expression and characterization of a novel human anti-IgE scFv fragment

Antibodies highly specific to human immunoglobulin (Ig) E are capable of selectively blocking the IgE interaction or eliminating IgE-producing cells, thus providing valuable agents for diagnostics and treatment of various allergic illness. An example is omalizumab, a humanized monoclonal anti-IgE antibody that is approved for the treatment of patients with moderate-to-severe allergic diseases in the United States, European Union and other countries. Here, we describe the generation and characterization of a novel human anti-IgE as a single-chain antibody fragment (scFv). The bacterially-synthesized scFv showed high affinity (86 nM) and specificity to the Fc region of human IgE. To our knowledge, this is the first report of the production of a human anti-IgE scFv in E. coli. Its further development as a potential candidate for medical applications is discussed.