Recombinant antibodies provide an emerging strategy in the development of new immunosensors. In p... more Recombinant antibodies provide an emerging strategy in the development of new immunosensors. In particular, single chain antibodies (scFvs) can be isolated and expressed in bacterial systems that also allow their in vitro manipulation at the gene level. In this work, we present for the first time results of single-chain phage displayed antibodies combined with amperometric detection and its application as an immunosensor. The scFv is immobilized on a carbon electrode and used to capture and quantify its specific target antigen. We describe the detection of the sugar milk lactose, the bacteria Listeria monocytogenes, and the enzyme MtKatG, which is expressed by Mycobacteriumtuberculosis.
The mAb B1 (mouse IgG1 kappa) recognizes a carbohydrate epitope on human carcinoma cells (I. Past... more The mAb B1 (mouse IgG1 kappa) recognizes a carbohydrate epitope on human carcinoma cells (I. Pastan et al., Cancer Res., 51: 3781-3787, 1991). We have generated plasmids encoding immunotoxins in which the Fv domain of B1, either as a single-chain Fv or as a disulfide-stabilized Fv (dsFv), was fused to PE38, a truncated form of Pseudomonas exotoxin A. To compare the activities of the two types of recombinant immunotoxins, the proteins were prepared from cytoplasmic inclusion bodies produced in Escherichia coli. The immunotoxins were evaluated for stability, antigen binding, specific cytotoxicity, pharmacokinetics, and antitumor activity in a nude mouse model. Although the single-chain immunotoxin is relatively stable when incubated at 37 degreesC (t(1/2) approximately 4 h), the dsFv immunotoxin is much more stable, with no loss of activity after 8 h at 37 degreesC. The single-chain immunotoxin has a 2-fold better binding affinity and cytotoxicity toward antigen-positive cultured cells than the dsFv immunotoxin. The half-lives in the blood of mice of B1(Fv)PE38 (single-chain) and B1(dsFv)PE38 (disulfide-stabilized) are 23 and 27 min, respectively. Their therapeutic potential was evaluated in athymic nude mice bearing human epidermoid carcinoma xenografts. Both immunotoxins caused complete regressions of the s.c. (30-40 mm3) tumors when given i.v. in three doses of 0.025 mg/kg every other day. This is one-twentieth of the mouse LD50. Recombinant immunotoxins containing the B1(Fv) are 2-3-fold more potent antitumor agents than previously described immunotoxins containing the B3(Fv) (Brinkmann et al., Proc. Natl. Acad. Sci. USA, 88: 8616-8620, 1991), which also target LeY and related carbohydrates in human tumors, but have a similar toxicity in mice. Thus, their therapeutic window is 2-3-fold larger. In addition, B1(dsFv)PE38 has only a 50% decrease in the apparent binding affinity of B1(Fv)PE38, whereas B3(dsFv)PE38 has a much greater loss in antigen binding.
Pseudomonas exotoxin A (PE) is a three-domain protein in which domain Ia is involved in recogniti... more Pseudomonas exotoxin A (PE) is a three-domain protein in which domain Ia is involved in recognition of receptors on eukaryotic target cells, domain II promotes translocation of PE into the cytosol, and domain III enzymatically ADP-ribosylates elongation factor 2. Modification of proteins with polyethylene glycol (PEG) has been shown to prolong circulating plasma lifetime and may reduce or eliminate immunogenicity. However, in the case of toxins, PEG may interfere with or block toxin activity. To investigate the effect of polyethylene glycolation on specific residues located on the surface of PE domain II, we substituted cysteine, for each of the five most exposed surface amino acids (H276, E282, N306, R313, and E327) in domain II. These cysteines can serve as unique sites for PEG modification. The PE-Cys proteins retained most of their cytotoxicity even when the free sulfhydryl group was blocked by 5,5'-dithiobis(nitrobenzoic acid) or glutathione. When the PE-Cys proteins were conjugated with ovalbumin using a cleavable disulfide linkage, cytotoxicity was retained, but it was lost with a non-cleavable thioether linkage. In contrast, cytotoxicity was maintained when PE-Cys mutants were coupled to 5- or 20-kDa mPEG, using either a disulfide or a thioether linkage. Unexpectedly in some cases, the thioether conjugate was more active than the disulfide linkage. Pharmacokinetic studies on one of the polyethylene-glycolated molecules (R313C) showed that the mean residence time (t 1/2) was prolonged to 72 min, compared to 20 min for unpolyethylene glycolated PE-Cys(R313C). These studies show it is possible to derivatize PE at specific residues in domain II, maintain significant cytotoxic activity, and alter pharmacokinetics. These studies also suggest that large mPEG molecules can be translocated to the cytosol while still attached to domain II of PE.
Bispecific antibodies (bsAbs) are antibodies with two binding sites directed at different antigen... more Bispecific antibodies (bsAbs) are antibodies with two binding sites directed at different antigens, enabling therapeutic strategies not possible with conventional monoclonal antibodies (mAbs). Since bispecific antibodies are regarded as promising therapeutic agents, many different bispecific design modalities have been evaluated. Many of these are based on antibody fragments or on inclusion of non-antibody components. For some therapeutic applications, full-size, native IgG-like bsAbs may be the optimal format.To prepare bsAbs in IgG format, two challenges should be met. One is that each heavy chain will only pair with the heavy chain of the second specificity and that heavy chain homodimerization will be prevented. The second is that each heavy chain will only pair with the light chain of its own specificity and that pairing with the light chain of the second specificity will be prevented. The first solution to the first criterion (known as knobs into holes, KIH) was presented in 1996 by Genentech and additional solutions were presented more recently. However, until recently, out of >120 published formats, only a handful of solutions for the second criterion that make it possible to produce a bispecific IgG by a single expressing cell were suggested.Here, we present a protocol for preparing bsAbs in IgG format in transfected mammalian cells. For heavy chain dimerization we use KIH while as a solution for the second challenge-correct pairing of heavy and light chains of bispecific IgGs we present our "BIClonals" technology; an engineered (artificial) disulfide bond between the antibodies' variable domains that asymmetrically replaces the natural disulfide bond between CH1 and CL.During our studies of bsAbs we found that H-L chain pairing seems to be driven by VH-VL interfacial interactions that differ between different antibodies; hence, there is no single optimal solution for effective and precise assembly of bispecific IgGs that suits every antibody sequence, making it necessary to carefully evaluate the optimal solution for each new antibody.
BackgroundEosinophilic esophagitis (EoE) is a chronic, food‐driven allergic disease, characterize... more BackgroundEosinophilic esophagitis (EoE) is a chronic, food‐driven allergic disease, characterized by eosinophil‐rich inflammation in the esophagus. The histopathological and clinical features of EoE have been attributed to overproduction of the type 2 cytokines IL‐4 and IL‐13, which mediate profound alterations in the esophageal epithelium and neutralizing of their shared receptor component (IL‐4Rα) with a human antibody drug (dupilumab) demonstrates clinical efficacy. Yet, the relative contribution of IL‐4 and IL‐13 and whether the type II IL‐4 receptor (comprised of the IL‐4Rα chain in association with IL‐13Rα1) mediates this effect has not been determined.MethodsExperimental EoE was induced in WT, Il13ra1−/−, and Krt14Cre/Il13ra1fl/fl mice by skin‐sensitized using 4‐ethoxymethylene‐2‐phenyl‐2‐oxazolin (OXA) followed by intraesophageal challenges. Esophageal histopathology was determined histologically. RNA was extracted and sequenced for transcriptome analysis and compared with ...
Recombinant antibodies provide an emerging strategy in the development of new immunosensors. In p... more Recombinant antibodies provide an emerging strategy in the development of new immunosensors. In particular, single chain antibodies (scFvs) can be isolated and expressed in bacterial systems that also allow their in vitro manipulation at the gene level. In this work, we present for the first time results of single-chain phage displayed antibodies combined with amperometric detection and its application as an immunosensor. The scFv is immobilized on a carbon electrode and used to capture and quantify its specific target antigen. We describe the detection of the sugar milk lactose, the bacteria Listeria monocytogenes, and the enzyme MtKatG, which is expressed by Mycobacteriumtuberculosis.
The mAb B1 (mouse IgG1 kappa) recognizes a carbohydrate epitope on human carcinoma cells (I. Past... more The mAb B1 (mouse IgG1 kappa) recognizes a carbohydrate epitope on human carcinoma cells (I. Pastan et al., Cancer Res., 51: 3781-3787, 1991). We have generated plasmids encoding immunotoxins in which the Fv domain of B1, either as a single-chain Fv or as a disulfide-stabilized Fv (dsFv), was fused to PE38, a truncated form of Pseudomonas exotoxin A. To compare the activities of the two types of recombinant immunotoxins, the proteins were prepared from cytoplasmic inclusion bodies produced in Escherichia coli. The immunotoxins were evaluated for stability, antigen binding, specific cytotoxicity, pharmacokinetics, and antitumor activity in a nude mouse model. Although the single-chain immunotoxin is relatively stable when incubated at 37 degreesC (t(1/2) approximately 4 h), the dsFv immunotoxin is much more stable, with no loss of activity after 8 h at 37 degreesC. The single-chain immunotoxin has a 2-fold better binding affinity and cytotoxicity toward antigen-positive cultured cells than the dsFv immunotoxin. The half-lives in the blood of mice of B1(Fv)PE38 (single-chain) and B1(dsFv)PE38 (disulfide-stabilized) are 23 and 27 min, respectively. Their therapeutic potential was evaluated in athymic nude mice bearing human epidermoid carcinoma xenografts. Both immunotoxins caused complete regressions of the s.c. (30-40 mm3) tumors when given i.v. in three doses of 0.025 mg/kg every other day. This is one-twentieth of the mouse LD50. Recombinant immunotoxins containing the B1(Fv) are 2-3-fold more potent antitumor agents than previously described immunotoxins containing the B3(Fv) (Brinkmann et al., Proc. Natl. Acad. Sci. USA, 88: 8616-8620, 1991), which also target LeY and related carbohydrates in human tumors, but have a similar toxicity in mice. Thus, their therapeutic window is 2-3-fold larger. In addition, B1(dsFv)PE38 has only a 50% decrease in the apparent binding affinity of B1(Fv)PE38, whereas B3(dsFv)PE38 has a much greater loss in antigen binding.
Pseudomonas exotoxin A (PE) is a three-domain protein in which domain Ia is involved in recogniti... more Pseudomonas exotoxin A (PE) is a three-domain protein in which domain Ia is involved in recognition of receptors on eukaryotic target cells, domain II promotes translocation of PE into the cytosol, and domain III enzymatically ADP-ribosylates elongation factor 2. Modification of proteins with polyethylene glycol (PEG) has been shown to prolong circulating plasma lifetime and may reduce or eliminate immunogenicity. However, in the case of toxins, PEG may interfere with or block toxin activity. To investigate the effect of polyethylene glycolation on specific residues located on the surface of PE domain II, we substituted cysteine, for each of the five most exposed surface amino acids (H276, E282, N306, R313, and E327) in domain II. These cysteines can serve as unique sites for PEG modification. The PE-Cys proteins retained most of their cytotoxicity even when the free sulfhydryl group was blocked by 5,5'-dithiobis(nitrobenzoic acid) or glutathione. When the PE-Cys proteins were conjugated with ovalbumin using a cleavable disulfide linkage, cytotoxicity was retained, but it was lost with a non-cleavable thioether linkage. In contrast, cytotoxicity was maintained when PE-Cys mutants were coupled to 5- or 20-kDa mPEG, using either a disulfide or a thioether linkage. Unexpectedly in some cases, the thioether conjugate was more active than the disulfide linkage. Pharmacokinetic studies on one of the polyethylene-glycolated molecules (R313C) showed that the mean residence time (t 1/2) was prolonged to 72 min, compared to 20 min for unpolyethylene glycolated PE-Cys(R313C). These studies show it is possible to derivatize PE at specific residues in domain II, maintain significant cytotoxic activity, and alter pharmacokinetics. These studies also suggest that large mPEG molecules can be translocated to the cytosol while still attached to domain II of PE.
Bispecific antibodies (bsAbs) are antibodies with two binding sites directed at different antigen... more Bispecific antibodies (bsAbs) are antibodies with two binding sites directed at different antigens, enabling therapeutic strategies not possible with conventional monoclonal antibodies (mAbs). Since bispecific antibodies are regarded as promising therapeutic agents, many different bispecific design modalities have been evaluated. Many of these are based on antibody fragments or on inclusion of non-antibody components. For some therapeutic applications, full-size, native IgG-like bsAbs may be the optimal format.To prepare bsAbs in IgG format, two challenges should be met. One is that each heavy chain will only pair with the heavy chain of the second specificity and that heavy chain homodimerization will be prevented. The second is that each heavy chain will only pair with the light chain of its own specificity and that pairing with the light chain of the second specificity will be prevented. The first solution to the first criterion (known as knobs into holes, KIH) was presented in 1996 by Genentech and additional solutions were presented more recently. However, until recently, out of >120 published formats, only a handful of solutions for the second criterion that make it possible to produce a bispecific IgG by a single expressing cell were suggested.Here, we present a protocol for preparing bsAbs in IgG format in transfected mammalian cells. For heavy chain dimerization we use KIH while as a solution for the second challenge-correct pairing of heavy and light chains of bispecific IgGs we present our "BIClonals" technology; an engineered (artificial) disulfide bond between the antibodies' variable domains that asymmetrically replaces the natural disulfide bond between CH1 and CL.During our studies of bsAbs we found that H-L chain pairing seems to be driven by VH-VL interfacial interactions that differ between different antibodies; hence, there is no single optimal solution for effective and precise assembly of bispecific IgGs that suits every antibody sequence, making it necessary to carefully evaluate the optimal solution for each new antibody.
BackgroundEosinophilic esophagitis (EoE) is a chronic, food‐driven allergic disease, characterize... more BackgroundEosinophilic esophagitis (EoE) is a chronic, food‐driven allergic disease, characterized by eosinophil‐rich inflammation in the esophagus. The histopathological and clinical features of EoE have been attributed to overproduction of the type 2 cytokines IL‐4 and IL‐13, which mediate profound alterations in the esophageal epithelium and neutralizing of their shared receptor component (IL‐4Rα) with a human antibody drug (dupilumab) demonstrates clinical efficacy. Yet, the relative contribution of IL‐4 and IL‐13 and whether the type II IL‐4 receptor (comprised of the IL‐4Rα chain in association with IL‐13Rα1) mediates this effect has not been determined.MethodsExperimental EoE was induced in WT, Il13ra1−/−, and Krt14Cre/Il13ra1fl/fl mice by skin‐sensitized using 4‐ethoxymethylene‐2‐phenyl‐2‐oxazolin (OXA) followed by intraesophageal challenges. Esophageal histopathology was determined histologically. RNA was extracted and sequenced for transcriptome analysis and compared with ...
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Papers by Itai Benhar