Immunotoxins of Pseudomonas exotoxin A (PE): effect of linkage on conjugate yield, potency, selectivity and toxicity

Conjugates of monoclonal antibodies and Pseudomonas exotoxin A (PE) were formed with disulfide or thioether bonds. Thioether conjugates which formed with succinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC) modified PE and reduced antibody formed with an 80% yield of equimolar conjugate within 30 min with an offering of one to one (toxin:antibody). The efficiency and kinetics of thioether formation were much higher with SMCC than with other maleimide reagents as well as more efficient than disulfide linkers. Thioether linkage resulted in immunotoxin consistently more potent and more selective in vitro than disulfide bonded conjugate. Thioether bonded conjugates also proved to have other favorable in vivo properties compared to disulfide conjugates: (1) a longer half-life in serum; (2) increased tumor localization; and (3) reduced toxicity. Toxicity of thioether linked holotoxin conjugates was directed at the liver hepatocyte but was easily monitored by serum liver enzymes. The conjugates are currently undergoing clinical evaluation for treatment of ovarian cancer with intraperitoneal administration. Research is ongoing to further decrease residual toxicity without reducing the potency of the conjugate.     

Preparation and characterization of copper-67 porphyrin-antibody conjugates

Methods were developed to label antibodies with copper-67, a potentially useful medical radioisotope, using the porphyrin chelating agent N-benzyl-5,10,15,20-tetrakis(4-carboxyphenyl) porphine. The porphyrin was activated for coupling using either (1) 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide HCl and N-hydroxysuccinimide or (2) 1,1'-carbonyldiimidazole. The coupling reactions were optimized as a function of activation time, coupling time, coupling pH, and reagent concentrations to achieve maximum coupling to IgG monomer. Sodium dodecylsulfate polyacrylamide gel electrophoresis was used to determine coupling yields. After purification by gel filtration, the antibody-porphyrin conjugates were labeled with copper-67 in aqueous solution. The coupling protocols were used to label antibodies from several species, demonstrating the general utility of these methods. Characterization of the conjugates indicated that the porphyrin label was attached randomly to the IgG molecule. Antigen binding capacities after conjugation were unaltered or slightly lowered as determined by a competitive ELISA.     

Synthetic peptides as antigens: pitfalls of conjugation methods

Peptide-carrier conjugates were prepared using 9 different synthetic peptides, 3 carrier proteins and 4 coupling reagents. Residues of the carrier protein that were modified by different coupling reagents (e.g., glutaraldehyde, carbodiimides, bis-diazotized benzidine) were found to elicit specific antibodies that reacted with unrelated carrier proteins treated with the same coupling agent. To demonstrate the presence of peptide antibodies in an antiserum raised against a peptide-carrier conjugate, it was necessary to use a antigen the peptide coupled to another carrier by means of a different coupling agent. Some of the commonly used conjugation methods were found to lead to conjugates of insufficient stability and sometimes also altered the antigenic properties of the peptide moiety. These difficulties can be overcome by additional control experiments designed to test the quality and the peptide-carrier conjugates.     

A comparison of mineral affinity of bisphosphonate-protein conjugates constructed with disulfide and thioether linkages

Chemical conjugation of bisphosphonates (BPs) to therapeutic proteins is an effective means to impart mineral affinity to proteins. Such conjugates can be implanted with mineral-based matrices to control the local delivery kinetics of the proteins. BPs linked to proteins with reversible (i.e., cleavable) linkages are desirable over conjugates with stable linkages to release the protein in free form. This study conducted a direct comparison of mineral affinity of BP-protein conjugates linked together with cleavable disulfide and non-cleavable thioether linkages. Bovine serum albumin (BSA) was used as a model protein and the desired conjugates were created with N-succinimidyl-3-(2-pyridyldithio)propionate (disulfide) and succinimidyl-4-(N-maleimido-methyl)cyclohexane-1-carboxylate (thioether) linkers. The disulfide-linked conjugates were cleaved in the presence of a major thiol constituent of serum, cysteine. The imparted mineral affinity, as assessed by hydroxyapatite binding in vitro, was lost upon the cleavage of the disulfide-linked aminoBP. The presence of the serum did not accelerate the cleavage of disulfide-linked conjugates. The aminoBP-BSA conjugates formed with disulfide and thioether linkages were subcutaneously implanted in rats with two different mineral-based matrices to assess protein loss from the matrices. All conjugates exhibited a higher retention in mineral matrices as compared to unmodified BSA. However, no significant differences in in situ pharmacokinetics of the disulfide- and thioether-linked conjugates were observed. We conclude that disulfide-linked BP conjugates were readily cleavable by the amino acid cysteine in vitro, but in vivo cleavage of the disulfide-linked conjugates was not evident when the proteins were implanted adsorbed to mineral-based matrices. BP-protein conjugates with faster-cleaving tethers might be required to significantly influence the release of the BP conjugates from the mineral matrices.     

Comparison of four bifunctional reagents for coupling peptides to proteins and the effect of the three moieties on the immunogenicity of the conjugates

Peptide-carrier conjugates are widely used to raise antipeptide antibodies. In a model system using angiotensin and tetanus toxoid as the peptide and the carrier protein respectively, four cross-linking reagents were employed to study their effect on the immunogenicity of the conjugates. Optimization of the conjugation method for these heterobifunctional reagents, all succinimidyl active esters, resulted in well-defined conjugates of predictable composition. ELISA assays were performed to compare the antigenicity and the immunogenicity of the conjugates. The antipeptide antibody titres were of the order of 2 X 10(4)-2 X 10(5). The anti-carrier antibody titres were high, in spite of the modification of the protein. Three of the four coupling reagents used for conjugation were of the 'maleimide' type: succinimidyl 6-(N-maleimido)-n-hexanoate (MHS), succinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC) and succinimidyl m-maleimidobenzoate (MBS). One coupling reagent contained an activated disulphide: succinimidyl 3-(2-pyridyldithio)propionate (SPDP). The constrained linkers originating from SMCC and MBS induced very high linker-specific antibody levels. The more flexible non-aromatic linkers originating from MHS and SPDP showed almost no reactivity. For this reason and since the thioether linkage is more stable than the disulphide bond, we recommend MHS as the crosslinking reagent of choice.     

The use of the fusion protein RGD-HSA-TIMP2 as a tumor targeting imaging probe for SPECT and PET

The human serum albumin tissue inhibitor of metalloproteinase 2 (HSA-TIMP2) is known to possess antitumor activity, which has been attributed to its ability to inhibit endothelial cell proliferation by binding to integrin receptors. In this study, a fusion protein, cyclic arginine-glycine-aspartate (RGD)-HSA-TIMP2, formed by conjugating HSA-TIMP2 with a RGD peptide, and its (123)I- and (68)Ga-labeled compounds, were synthesized and evaluated with in vivo tumor imaging using single photon emission computed tomography (SPECT) and positron emission tomography (PET). RGD-HSA-TIMP2 was synthesized by covalent bonding of the RGD peptide to the side chain amino groups of HSA-TIMP2 from a two-step reaction involving from activation with N-succinimidyl iodoacetate. This conjugation improved the anticancer effect of HSA-TIMP2 in cancer cells. The (123)I- and (68)Ga-labeled fusion proteins were prepared and subsequently injected into the tail veins of mice bearing human glioblastoma cancer U87MG xenografts for SPECT and PET imaging and biodistribution studies. Tumor uptake of radioligand was high in both the PET images and in the biodistribution studies at 3 h after injection. These studies demonstrated that the new fusion protein has potential not only as an anticancer agent but also as a radioligand for the diagnosis of tumors.     

Thermostable alpha-amylase conjugated antibodies as probes for immunodetection in ELISA

Thermostable alpha-amylase from B. licheniformis has been conjugated with high efficiency to goat antibodies against human, mouse, and rabbit immunoglobulins to prepare second-step reagents which can be used in Enzyme Linked Immunosorbent Assays (ELISA). Various conjugation methods, such as one- and two-step glutaraldehyde coupling and cross-linking, using heterobifunctional reagents such as sulfosuccinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carbonate (sulfo-SMCC) and N-succinimidyl-S-acetylthioacetate (SATA), yielded active alpha-amylase labeled second antibodies. Such conjugates had molecular sizes ranging between 200-300 kDa. Filter sterilized solutions of conjugates, when stored at 37 degrees C for two weeks, retained 32% of their biological activity and were thermostable even after keeping for 1 h at 90 degrees C.     

A comparison of different enzyme-antibody conjugates for enzyme-linked immunosorbent assay.

Calf intestinal alkaline phosphatase (CIAP)- and horseradish peroxidase (HRP)-linked goat antibody (Ab) conjugates have been prepared by two procedures. One procedure is by glutaraldehyde coupling of the proteins; the other is by intermolecular disulfide bond formation of the appropriately modified proteins. The conjugates, specific for rabbit IgG, were tested for their effectiveness as reagents in enzyme-linked immunosorbent assays for ragweed antigen E specific rabbit antibody. The CIAP-Ab conjugate prepared by glutaraldehyde coupling and the HRP-Ab conjugate made by disulfide bond formation were equally effective reagents for immunoassay, but the HRP-Ab conjugate obtained by glutaraldehyde coupling was definitely less useful than the other two conjugates. The assay procedure utilized an antigen E coupled paper disc as the immunosorbent and it was sensitive in the range of 0.5--100 ng per test. Inhibition of the enzyme-linked immunosorbent assay was used as a sensitive technique for measuring antigenic activity of antigen E or its derivatives.     

Thiol-reactive compounds prevent nonspecific antibody binding in immunohistochemistry

Nonspecific antibody binding is the primary source of confounding background in immunohistochemistry (IHC). Based on observed patterns of background staining, and the known spontaneous reduction of immunoglobulin disulfide bonds in vivo and in vitro, we tested the hypothesis that nonspecific antibody binding in IHC is mediated by sulfhydryl interactions. Coincubation of primary antibodies with reduced glutathione (GSH), L-cysteine, iodoacetic acid, Ellman's reagent and other thiophilic reagents in pH 8 tris-EDTA (TE) buffer inhibited background staining. In contrast, oxidized glutathione (GSSG) exerted no effect. When empirically optimized, coincubation of GSH with primary antibodies significantly improved IHC signal:noise ratio. Tissue preincubation with mercaptans, soft and borderline metals, and other sulfhydryl-reactive reagents also inhibited background staining, but at the expense of target sensitivity. ELISA results confirmed direct binding between murine serum antibodies and GSH in a nonantigen-dependent manner. In summary, thiol-reactive compounds prevent nonspecific antibody binding in IHC. We propose a mechanism whereby nonspecific background resulting from formation of disulfide bridges and other sulfhydryl bonds between primary antibodies and tissue side groups is interrupted by prior exposure to thiol-reactive reagents such as GSH. These findings provide a molecular basis to improve the specificity of IHC and other immunoassays, and hold implications for antibody-based clinical diagnostics and therapeutics.     

A novel carbodiimide coupling method for synthetic peptides. Enhanced anti-peptide antibody responses

Coupling of peptides to immunogenic protein carriers is required for the generation of anti-peptide antibody responses. Carbodiimides are hetero bi-functional coupling reagents that are utilized for coupling reactions through carboxyl and amino groups. The procedures generally used for carbodiimide coupling of peptides and proteins result in conjugates which generate immunodominant antibody responses directed against the neodeterminants on the carrier protein. These determinants are induced by the reaction of carrier and/or peptide with the coupling agent. We have investigated the potential inhibiting effect of an imidazole intermediary on the formation of unwanted neodeterminants during carbodiimide coupling. The serum antibody responses elicited with the peptide-protein conjugates produced were evaluated in ELISA. We have modified and improved the coupling with a watersoluble carbodiimide (EDC) in such a way that a high response to the coupled peptide is obtained in association with negligible levels of anti-neodeterminant antibodies.     

THERMOSTABLE α-AMYLASE CONJUGATED ANTIBODIES AS PROBES FOR IMMUNODETECTION IN ELISA

ABSTRACT

Thermostable α-amylase from B. licheniformis has been conjugated with high efficiency to goat antibodies against human, mouse, and rabbit immunoglobulins to prepare second-step reagents which can be used in Enzyme Linked Immunosorbent Assays (ELISA). Various conjugation methods, such as one- and two-step glutaraldehyde coupling and cross-linking, using heterobifunctional reagents such as sulfosuccinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carbonate (sulfo-SMCC) and N-succinimidyl-S-acetylthioacetate (SATA), yielded active α-amylase labeled second antibodies. Such conjugates had molecular sizes ranging between 200–300 kDa. Filter sterilized solutions of conjugates, when stored at 37°C for two weeks, retained 32% of their biological activity and were thermostable even after keeping for 1 h at 90°C.     

Mono- and tri-cationic porphyrin-monoclonal antibody conjugates: photodynamic activity and mechanism of action

Two cationic porphyrins bearing an isothiocyanate group for conjugation to monocolonal antibodies have been synthesized. The two porphyrins conjugated efficiently to three monoclonal antibodies (anti-CD104, anti-CD146 and anti-CD326), which recognize antigens commonly over-expressed on a range of tumour cells. In vitro, all conjugates retained the phototoxicity of the porphyrin and the immunoreactivity of the antibody. Mechanistic studies showed that conjugates formed from the mono- and tri-cationic porphyrin and anti-CD104 antibody mediated apoptosis following irradiation with non-thermal red light of 630 ± 15 nm wavelength. In vivo antibody conjugates caused suppression of human LoVo tumour growth in immunodeficient NIH III mice, similar to the commercial photodynamic therapy (PDT) agent Photofrin, but at administered photosensitizer doses that were more than two orders of magnitude lower. Positron emission tomography (PET) following PDT showed a large, early increase in uptake of (18) fluorodeoxyglucose (FDG) by tumours treated with the anti-CD104 conjugates. This effect was not observed with Photofrin or with conjugates formed from the same photosensitizers conjugated to an irrelevant antibody.     

Anti-human interleukin 2 receptor monoclonal antibody isotypic switching: Chimeric rat-human antibodies

Univalent and bivalent (2Fab'-Fc) chimeric rat-human antibodies were constructed by chemical coupling (thioether bonds) of Fab' fragments from the 33B31 rat anti-human interleukin 2 receptor ( chain) monoclonal antibody to human IgG or Fc fragments. The purity of the chimeric antibodies obtained after purification was assessed by the sodium dodecyl sulfide-polyacrylamide gel electrophoresis method. The affinity of chimeric antibodies for the human interleukin 2 receptor was determined. The affinity of the 2Fab'-Fc was better (Kd = 0.87 nM) than that of the Fab'-IgG (Kd = 2.04 nM) or the Fab'-Fc chimera (Kd = 3.93 nM). Binding studies on a T-cell clone showed that the percentage of positive cells recognized by chimeric antibodies was comparable to that obtained with unmodified 33B3.1 IgG2a or its corresponding Fab' fragment. In addition, the inhibition of interleukin 2-induced cell proliferation and allogeneic proliferative response by chimeric antibodies was of the same magnitude as that obtained with the rat IgG2a anti-interleukin 2 receptor monoclonal antibody and Fab' fragments. This study shows the possibility of changing the isotype of monoclonal antibodies without important modification to their binding activity. These reagents may offer an alternative to unmodified monoclonal antibodies for therapeutic application.     

Direct fluorochrome labeling of phage display library clones for studying binding specificities: applications in flow cytometry and fluorescence microscopy

Phage display technology is increasingly employed to identify high-affinity peptides and single-chain antibodies with binding specificities for a diversity of target types. The analysis of phage-binding sensitivity and specificity typically employs directly labeled secondary antiphage antibodies and potentially tertiary labels, such as fluorochromes and enzymes, when biotinylated antibodies are used. However, secondary or tertiary reagents may not be feasible or desirable for some target types and applications. Here, we present a simple approach for directly labeling phage clones with two common amine-reactive fluorochromes. We show that these fluorochromes label the pVIII major coat protein and that the binding selectivity of peptides displayed on the pIII protein of several well-characterized phage clones is maintained in flow cytometric analysis and immunofluorescence microscopy. Uniquely, such labeled phage, in part, represent self-propagating reagents because conjugation does not impair the ability to efficiently reproduce in bacteria, although relabeling with fluorochrome would be necessary. Our data suggest that primary labeled phage clones may be used similarly to primary antibody conjugates.     

125 I标记的抗p185抗体的体外代谢与体内生物学分布

目的：研究^125Ⅰ标记的抗p185抗体（鼠mAb A21、人鼠嵌合抗体A21 scFv-Fc及单链抗体A21 seFv）在体外的细胞代谢和在体内的生物学分布，为其在诊断和治疗高表达p185肿瘤的临床应用提供依据。方法：通过FACS检测抗体是否与高表达膜表面抗原p185的SKOV，特异结合。采用氯胺T法进行碘标记抗体，用细胞放射免疫分析实验，检测抗体在SKOV、细胞中的代谢。建立荷SKOV，裸鼠模型，以观察抗体的体内生物学分布。结果：体外细胞代谢实验表明，抗体在与细胞膜表面结合后，进而被内吞入细胞，在细胞内降解和脱碘，最后被分泌出细胞外。体内药物代谢实验表明，抗体在动物体内肿瘤部位出现选择性放射性浓聚，而无关抗体未出现放射性浓聚，呈全身分布。结论：mAb A21，A21 seFv-Fc和A21 scFv对于高表达p185的卵巢癌在体内和体外都具有亲和力，可单用作高表挟D185肿瘤的诊断和治疗。     

~(125)I标记的抗p185抗体的体外代谢与体内生物学分布

目的:研究125I标记的抗p185抗体(鼠mAbA21、人鼠嵌合抗体A21scFv-Fc及单链抗体A21scFv)在体外的细胞代谢和在体内的生物学分布,为其在诊断和治疗高表达p185肿瘤的临床应用提供依据。方法:通过FACS检测抗体是否与高表达膜表面抗原p185的SKOV3特异结合。采用氯胺T法进行碘标记抗体,用细胞放射免疫分析实验,检测抗体在SKOV3细胞中的代谢。建立荷SKOV3裸鼠模型,以观察抗体的体内生物学分布。结果:体外细胞代谢实验表明,抗体在与细胞膜表面结合后,进而被内吞入细胞,在细胞内降解和脱碘,最后被分泌出细胞外。体内药物代谢实验表明,抗体在动物体内肿瘤部位出现选择性放射性浓聚,而无关抗体未出现放射性浓聚,呈全身分布。结论:mAbA21,A21scFv-Fc和A21scFv对于高表达p185的卵巢癌在体内和体外都具有亲和力,可望用作高表达p185肿瘤的诊断和治疗。     

Folate coupled poly(ethyleneglycol) conjugates of anionic poly(amidoamine) dendrimer for inflammatory tissue specific drug delivery

Folate receptor is overexpressed on the activated (but not quiescent) macrophages in both animal models and human patients with naturally occurring rheumatoid arthritis. The aim of this study was to prepare folate targeted poly(ethylene glycol) (PEG) conjugates of anionic dendrimer (G3.5 PAMAM) as targeted drug delivery systems to inflammation and to investigate its biodistribution pattern in arthritic rats. Folate-PEG-PAMAM conjugates, with different degrees of substitution were synthesized by a two-step reaction through a carbodiimide-mediated coupling reaction and loaded with indomethacin. Folate-PEG conjugation increased the drug loading efficiency by 10- to 20-fold and the in vitro release profile indicated controlled release of drug. The plasma pharmacokinetic parameters indicated an increased AUC, circulatory half-life and mean residence time for the folate-PEG conjugates. The tissue distribution studies revealed significantly lesser uptake by stomach for the folate-PEG conjugates, thereby limiting gastric-related side effect. The time-averaged relative drug exposure (r(e)) of the drug in paw for the folate-PEG conjugates ranged from 1.81 to 2.37. The overall drug targeting efficiency (T(e)) was highest for folate-PEG conjugate (3.44) when compared to native dendrimer (1.72). The folate-PEG-PAMAM conjugates are the ideal choice for targeted delivery of antiarthritic drugs to inflammation with reduced side-effects and higher targeting efficiency.     

Folate receptor targeted imaging using poly (ethylene glycol)-folate: in vitro and in vivo studies

The aim of this study was to ascertain the folate receptor (FR) targetability by an in vitro study and to acquire FR-targeted images in vivo models by using synthetic folate conjugates. PEG-folate was synthesized and labeled with (99m)Tc and fluorescein isothiocynate (FITC). Cell uptake studies were carried out in KB cells (FR-positive) and A549 cells (FR-negative) using FITC- and the (99m)Tc-labeled conjugates. The radiolabeled conjugate was intravenously injected to KB tumor xenografted mice. After it was injected, gamma images were recorded at 30 min, 1, 2, 3 and 4 hr. Cell uptake studies showed a difference between the KB cells and the A549 cells by flow cytometry analysis and gamma counting. On in vivo images, the tumor-to-normal muscle ratio was greater than 4. It ascertained that the PEG-folate conjugate specifically binds to the FR expressed on tumor cells in vitro. Moreover, it was possible to acquire the FR-targeted gamma images using PEG-folate conjugates in tumor models.     

Biophysical and biochemical studies of the hair in cartilage-hair hypoplasia

The hair from four patients with classical cartilage-hair hypoplasia (CHH) was studied with respect to its stress-strain characteristics, amino acid composition, solubility in 6 M urea containing reducing agents, and the electrophoresis of the isolated S-carboxymethylated proteins in acrylamide gels containing 6 M urea. Many CHH hairs had the mechanical properties of hairs with reduced disulfide bonds and these properties returned to normal after five minutes' oxidation with 3 % hydrogen peroxide, There was decreased solubility of CHH hair with a deficiency of the filamenlous proteins when 0.1 M mercaptocthanol was the reducing agent; with 0.1 M dithiothreitol as reducing agent, the yield of proteins increased and the electrophoretic pattern was normal. The filamentous and matrix proteins of CHH hair have no gross structural defects, and it is suggested that the decreased reactivity of some disulfide bonds may he responsible for the abnormal biophysical and biochemical characteristics of this hair.     

Texas Red, a hydrophilic, red-emitting fluorophore for use with fluorescein in dual parameter flow microfluorometric and fluorescence microscopic studies

The sulfonylchloride derivative of the red-emitting fluorophore, sulforhodamine 101, has been synthesized in order to provide a reagent for coupling to amino groups on proteins and other compounds, and it is now commercially available under the name 'Texas Red'. Texas Red conjugates of antibodies and other proteins have been prepared in high protein yields; these conjugates retained their biological activities and were strongly fluorescent. The excitation and emission spectra of Texas Red conjugates are widely separated from those of molecules labeled with fluorescein isothiocyanate. Texas Red is therefore an excellent reagent for use in single or dual label flow microfluorometric and fluorescent microscopic studies.