A highly efficient preparation of neoglycoconjugate vaccines using subcarriers that bear clustered carbohydrate antigens

A limited amount of spacer-equipped carbohydrate haptens was linked by reductive amination to a subcarrier, an oligopeptide containing 16 amino groups, to give a hapten-carrying subcarrier (HCS). It was then linked, via the remaining free amino groups, to chicken serum albumin (CSA) to give a cross-linked neoglycoconjugate bearing the haptens in the form of clusters. Alternatively, the same type of a glycoconjugate, but with higher conjugation efficiency, was obtained when HCS was treated successively with squaric acid diethyl ester and CSA.     

Impact of linker and conjugation chemistry on antigen binding,Fc receptor binding and thermal stability of model antibody-drug conjugates

Antibody-drug conjugates (ADCs) with biotin as a model cargo tethered to IgG1 mAbs via different linkers and conjugation methods were prepared and tested for thermostability and ability to bind target antigen and Fc receptor. Most conjugates demonstrated decreased thermostability relative to unconjugated antibody, based on DSC, with carbohydrate and amine coupled ADCs showing the least effect compared with thiol coupled conjugates. A strong correlation between biotin-load and loss of stability is observed with thiol conjugation to one IgG scaffold, but the stability of a second IgG scaffold is relatively insensitive to biotin load. The same correlation for amine coupling was less significant. Binding of antibody to antigen and Fc receptor was investigated using surface plasmon resonance. None of the conjugates exhibited altered antigen affinity. Fc receptor FcγIIb (CD32b) interactions were investigated using captured antibody conjugate. Protein G and Protein A, known inhibitors of Fc receptor (FcR) binding to IgG, were also used to extend the analysis of the impact of conjugation on Fc receptor binding. H10_NPEG4 was the only conjugate to show significant negative impact to FcR binding, which is likely due to higher biotin-load compared with the other ADCs. The ADC aHIS_NLC and aHIS_TPEG8 demonstrated some loss in affinity for FcR, but to much lower extent. The general insensitivity of target binding and effector function of the IgG1 platform to conjugation highlight their utility. The observed changes in thermostability require consideration for the choice of conjugation chemistry, depending on the system being pursued and particular application of the conjugate.     

Reduction of antigenicity of Cry j I, major allergen of Japanese cedar pollen, by the attachment of polysaccharides

An attempt was made to mask the allergenic structure of a major allergen protein, Cry j I (CJI), in Japanese cedar pollen using the Maillard-type polysaccharide conjugation. The SDS-PAGE pattern of the CJI-galactomannan conjugate prepared by the Maillard reaction showed broad bands widely distributed from 50 kDa to more than 100 kDa, suggesting the attachment of galactomannan. The competitive enzyme-linked immunosorbent assay showed that the IgE antibody in the sera of cedar pollen-sensitive patients reacted strongly with CJI, while it did not react with the CJI-galactomannan conjugate. This result suggests that the antigenicity of CJI is greatly reduced by the conjugation with galactomannan.     

Production of polyclonal antibodies towards the immunodetection of insecticide phosalone

Summary Hapten synthesis for the production of specific insecticide phosalone polyclonal antibodies was carried out starting from an intermediate of the phosalone synthesis, 6-chloro-2-benzoxazolone1. Two haptens containing different spacers have been prepared: N-5-carboxypentyl-6-chloro-2-benzoxazolone7 and N-(2-oxo-3-aza-5-carboxypentyl)-6-chloro-2-benzoxazolone12. Each of these two haptens conjugated to bovine serum albumine (BSA) was used to immunize four rabbits. Immunoassays of phosalone were performed with ELISA using solid-phase bound hapten thyroglobulin conjugate and horseradish peroxidase labelled goat antirabbit IgG. The more sensitive response was observed when the antiserum obtained from the rabbit immunized with the hapten-BSA conjugate containing the N-2-oxo-3-aza-5-carboxypentyl spacer was in competition with the same hapten coupled to thyroglobulin. An identical response was obtained under the same conditions when using benzoxazolone instead of phosalone as competitor, showing a good recognition of the specific aromatic part of the organophosphate insecticide phosalone. Reduction of coating conjugate concentration (from 2 to 0.05g/well) and also the use of heterologous coating protein instead of homologous did improve the sensitivity, resulting in a concentration of phosalone required to inhibit binding by 50% of 2 mg/l and a detection limit of 0.02 mg/l.     

A structurally diversified linker enhances the immune response to a small carbohydrate hapten

The tether employed to covalently attach β-mannan disaccharide glycoconjugates influences the specificity of rabbit antibodies that protect against Candida albicans. Two glycoconjugates containing (1?→?2)-β-mannan disaccharides linked to chicken serum albumin (CSA) either via a structurally uniform or via a stereodiversified spacer were prepared and evaluated in immunization trials in mice and rabbits. Immunization with conjugate vaccine possessing a structurally diversified linker induced higher IgG titers against Candida albicans cell wall phosphomannan than a conjugate with a structurally uniform linker. These results suggest that affinity maturation and the specific antibody response can be shifted towards recognition of the desired hapten by employing a linker with diversified configuration.     

Impact of linker and conjugation chemistry on antigen binding,Fc receptor binding and thermal stability of model antibody-drug conjugates

Antibody-drug conjugates (ADCs) with biotin as a model cargo tethered to IgG1 mAbs via different linkers and conjugation methods were prepared and tested for thermostability and ability to bind target antigen and Fc receptor. Most conjugates demonstrated decreased thermostability relative to unconjugated antibody, based on DSC, with carbohydrate and amine coupled ADCs showing the least effect compared with thiol coupled conjugates. A strong correlation between biotin-load and loss of stability is observed with thiol conjugation to one IgG scaffold, but the stability of a second IgG scaffold is relatively insensitive to biotin load. The same correlation for amine coupling was less significant. Binding of antibody to antigen and Fc receptor was investigated using surface plasmon resonance. None of the conjugates exhibited altered antigen affinity. Fc receptor FcγIIb (CD32b) interactions were investigated using captured antibody conjugate. Protein G and Protein A, known inhibitors of Fc receptor (FcR) binding to IgG, were also used to extend the analysis of the impact of conjugation on Fc receptor binding. H10_NPEG4 was the only conjugate to show significant negative impact to FcR binding, which is likely due to higher biotin-load compared with the other ADCs. The ADC aHIS_NLC and aHIS_TPEG8 demonstrated some loss in affinity for FcR, but to much lower extent. The general insensitivity of target binding and effector function of the IgG1 platform to conjugation highlight their utility. The observed changes in thermostability require consideration for the choice of conjugation chemistry, depending on the system being pursued and particular application of the conjugate.     

Preparation of glycoconjugates by dialkyl squarate chemistry revisited

The methyl 6-hydroxyhexanoyl glycoside of lactose was treated with each of 1,2-diaminoethane or hydrazine hydrate, and the corresponding amino amide 4 and acyl hydrazide 13, were treated with each of squaric acid dimethyl, diethyl, dibutyl, and didecyl esters. The monoesters were conjugated to bovine serum albumin (BSA) at different concentrations of hapten using 0.05 and 0.5M pH 9 borate buffer. Maximum loading was achieved faster, and the conjugation efficiency was higher, when the conjugation was conducted at higher concentrations of both hapten and buffer. Conjugations involving haptens 14-17 prepared from hydrazide 13 were generally slower and less efficient than those with compounds 5-8, which were made from amino amide 4. Maintaining pH 9 during conjugation was found to be the most important factor in ensuring that the conjugation was a fast, highly efficient, and reproducible process. When the pH of the conjugation mixture fell during the reaction, resulting in decreased reaction rate or even termination of the conjugation process, the normal course of the conjugation process could be restored by addition of buffer salts. Hydrolysis studies with monoesters formed from amino amide 4 under conjugation conditions showed that decyl ester 8 was the most stable and that the methyl compound 5 was the one most readily hydrolyzed. The stability of monoesters prepared from hydrazide 13 was similar and comparable to the decyl ester prepared from 4. No definite advantage was found for the use of any of the four dialkyl squarate reagents (methyl-, ethyl-, butyl-, and decyl-) for conversion of carbohydrate derivatives to species amenable for conjugation. Nevertheless, dimethyl squarate seemed to be the most convenient reagent because it is a crystalline, easy to handle, and commercially available material with very good reactivity.     

A nondestructive method for peptide bond conjugation of antigenic haptens to a diphtheria toxoid carrier, exemplified by two antisera specific to acetolactate synthase.

A method for the preparation of an activated protein carrier is described: Protein carboxyl groups are transformed into N-hydroxysulfosuccinimide esters, a structure that will react with primary amino groups under amide bond formation. Although the activated ester is unstable under aqueous conditions, a significant amount of hapten molecules can be bound covalently to the carrier under very mild conditions. Ligands can be peptides or other molecules possessing a primary amino group. The method avoids the risk of ligand polymerization and no derivatization of the ligand prior to conjugation is needed. Residual unreacted ligand molecules can therefore be recovered in their native form by size exclusion chromatography. The method was used to conjugate two synthetic sugar beet acetolactate synthetase (E.C. 4.1.3.18) peptides to diphtheria toxoid. Antibodies were raised against both of the conjugates. The specificity of these antibodies against sugar beet acetolactate synthetase was verified using immunoblotting, ELISA, and immunoprecipitation.     

Use of different hapten-protein conjugates immobilized on nitrocellulose to screen monoclonal antibodies to abscisic acid

The dot-immunobinding method for screening antibodies to proteins on sheets of nitrocellulose has been modified to allow monoclonal antibodies (McAb) to the hapten abscisic acid (ABA) to be screened. Several methods for conjugating ABA to proteins using new bifunctional coupling reagents, specific for hapten keto groups, are described. Hybridomas secreting McAb with a defined specificity for the hapten can be identified by screening supernatants against the carrier protein and other hapten-protein conjugates with different conjugation bridges or modified hapten structure. Inhibition of binding to conjugates by free hapten is used to determine the relative avidity of the McAb for free and bound hapten. All of these tests could be done with no more than about 50 microliter of antibody solution. Dot immunobinding is a useful alternative to radioimmunoassay for screening McAb to haptens.     

An improved method for the microscale preparation and characterization of hapten-protein conjugates: the use of cholesterol as a model for nonchromophore hydroxylated haptens

A minute amount (0.446 micromol) of cholesterol (Chol) was converted into an hemisuccinate derivative (Chol HS) using an excess of succinic anhydride. The optimal conditions for synthesis of Chol HS were explored by checkerboard experiments in which various succinic anhydride/Chol molar ratios ranging from 5:1 to 30:1 were assayed over a wide temperature range (50-85 degrees C) and for various incubation times (3-8 h). Total conversion was obtained at the higher reagent ratios, temperatures, and incubation times. Subsequently, this carboxylic derivative was first covalently linked to bovine serum albumin (BSA) then to various proteins (casein, ovalbumin, and hemocyanins) or to a synthetic homopolymer (poly-DL-Lysine) via a modified version of the mixed anhydride method of Erlanger, performed in a reversed micellar medium. The assessment of the number of haptenic groups per mole of BSA (epitope density) was achieved chromatographically by two methods according to a Chol standard curve established at 207 nm with linearity in the range 0-50 microg. These procedures involving an alkaline hydrolysis of a sample of either the conjugate (direct method) or the unreacted Chol HS (indirect method) yielded an acceptable level of agreement and concordant results in all cases. The influence of the activated hapten/BSA molar ratio on the coupling efficiency was investigated by the direct method within the range 10:1 to 250:1. Using the optimal conditions determined for Chol HS synthesis (a molar reagent ratio of 30:1 with incubation at 65 degrees C for 6 h) and for BSA haptenation (a 100-fold molar excess of activated hapten, with a carrier stock concentration of 5 mg/mL), epitope density of the conjugates lied between 23 and 27. By reacting the same amount of activated hapten ( approximately 216 microg) with identical amounts of various carriers (300 microg), conjugation efficiency was found similar on a microgram of Chol bound per milligram of carrier basis. This simple and reproducible conjugation and analysis procedures should provide a general method applicable to poorly available and weakly immunogenic haptens bearing hydroxyl groups such as polyether-type marine toxins.     

Theoretical models for predicting the effect of bridging group recognition and conjugate substitution on hapten enzyme immunoassay dose-response curves

Models for predicting the effect of immunological recognition of the bridge group on the dose-response curves obtained with heterogeneous hapten enzyme immunoassays are presented. Appropriate theoretical treatment shows that the greater affinity of antibodies toward the enzyme-labeled species than for the unlabeled hapten analyte results in assays with limited detection capabilities. This problem is compounded when enzyme conjugates possessing multiple haptens are used. In equilibrium type competitive arrangements, the concentrations of binder and labeled hapten may be optimized to some extent to improve assay performance. However, the results presented show that only when assays are performed in a sequential binding mode using carefully controlled timing of reagent incubations can the detection capabilities of the assays be fully maximized for analyte measurements. Unfortunately, it is also shown that such sequential binding approaches render the assays essentially nonselective. The effect of decreasing the affinity of the binder to the enzyme-labeled hapten relative to the unlabeled analyte by using heterologous conjugates in equilibrium arrangements is shown to improve detection capabilities but also at the expense of reduced selectivity. Suggestions for reagent concentrations and conjugate substitution (degree of conjugation), which provide optimized dose-response curves at a given ED50 value, are also presented as are proposals for using different binders which do not exhibit bridging group recognition.     

Carboxymethyl cellulose, a nonimmunogenic hapten carrier with tolerogenic properties.

This communication reports on the tolerogenic properties of carboxymethyl cellulose (CMC) as a nonimmunogenic carrier for 2.4 dinitrophenyl (DNP) and the benzylpenicilloyl determinant (BPO). Either normal or primed mice, given an optimal dose of 250 micrograms per animal of DNP CMC, when challenged with an immunogenic form of the hapten as early as 30 min or as late as 21 days thereafter were completely and specifically unresponsive to it. Experimental evidence suggests that this unresponsiveness is not due to suppressor cells. Furthermore, DNP CMC induces tolerance in vivo but fails to do so in vitro under conditions at which other tolerogenic carbohydrate hapten conjugates such as DNP-dextran do. This together with comparative studies of tolerance induction kinetics by DNP CMC and DNP-dextran in vivo led us to conclude that molecular properties other than the epitope density must be attributed to CMC's tolerogenic potential. CMC may also be used as a tolerogenic carrier for BPO with respect to IgE antibody production. Thus, normal or primed mice injected with the BPO CMC conjugate were found specifically unresponsive to a challenge with an immunogenic form of penicillin.     

Conjugation of folate via gelonin carbohydrate residues retains ribosomal-inactivating properties of the toxin and permits targeting to folate receptor positive cells

Conjugation of folate to proteins permits receptor-mediated endocytosis via the folate receptor (FR) and delivery of the conjugate into the cytoplasm of cells. Since many cancers up-regulate the FR it has enabled the targeting of toxins to tumor cells resulting in specific cell death. However, current conjugation methods rely on chemistries that can affect certain catalytic subunits, such as the A-chain of the plant toxin gelonin. As a result many folate-targeted toxins are a compromise between receptor/ligand interaction and toxin activity. We describe the first example of folate conjugated to a protein via carbohydrate residues, using a novel SH-folate intermediate. The folate-gelonin conjugate retains over 99% of toxin activity in a cell-free translational assay compared with unmodified gelonin and is able to bind the FR at the same affinity as free folic acid (10(-10) m). Additionally, the conjugate exhibits prolonged inhibition of protein synthesis in FR positive cell lines in vitro. Folate linked to gelonin via amino conjugation exhibits the same affinity for FR as free folic acid but the toxin is 225-fold less active in a cell-free translational assay. The effect of different conjugation methods on toxin activity and the implications for folate targeting of other glycoproteins are discussed.     

Preclinical manufacture of an anti-HER2 scFv-PEG-DSPE, liposome-inserting conjugate. 1. Gram-scale production and purification

A GMP-compliant process is described for producing F5cys-PEG-lipid conjugate. This material fuses with preformed, drug-loaded liposomes, to form "immunoliposomes" that bind to HER2/neu overexpressing carcinomas, stimulates drug internalization, and ideally improves the encapsulated drug's therapeutic index. The soluble, single-chain, variable region antibody fragment, designated F5cys, was produced in E. coli strain RV308 using high-density cultures. Affinity adsorption onto horizontally tumbled Streamline rProtein-A resin robustly recovered F5cys from high-pressure-disrupted, whole-cell homogenates. Two product-related impurity classes were identified: F5cys with mid-sequence discontinuities and F5cys with remnants of a pelB leader peptide. Low-pressure cation exchange chromatography, conducted at elevated pH under reducing conditions, enriched target F5cys relative to these impurities and prepared a C-terminal cysteine for conjugation. Site-directed conjugation, conducted at pH 5.9 +/- 0.1 with reaction monitoring and cysteine quenching, yielded F5cys-MP-PEG(2000)-DSPE. Low-pressure size exclusion chromatography separated spontaneously formed, high-molecular-weight conjugate micelles from low-molecular-weight impurities. When formulated at 1-2 mg/mL in 10 mM trisodium citrate, 10% sucrose (w/v), at pH 6.4 (HCl), the conjugate was stable when stored below -70 degrees C. Six scale-up lots were compared. The largest 40-L culture produced enough F5cys to manufacture 2,085 mg of conjugate, enough to support planned preclinical and future clinical trials. The conjugate was 93% pure, as measured by polyacrylamide gel electrophoresis. Impurities were primarily identified as product-related. Residual endotoxin, rProtein A, and genomic DNA, were at acceptable levels. This study successfully addressed a necessary step in the scale-up of immunoliposome-encapsulated therapeutics.     

One-pot preparation of a series of glycoconjugates with predetermined antigen-carrier ratio from oligosaccharides that mimic the O-PS of Vibrio cholerae O:1, serotype Ogawa

Di-through the pentasaccharide that mimic the upstream terminus of the O-specific polysaccharide of Vibrio cholerae O:1, serotype Ogawa were synthesized in the form of 5-methoxycarbonylpentyl glycosides and linked to BSA using squaric acid diester chemistry. The conjugation reactions were monitored by surface-enhanced laser-desorption/ionization-time-of-flight mass spectrometry (SELDI-TOF MS), which allowed conducting the conjugation of the synthetic oligosaccharides in a controlled way and termination of the reaction when the desired molar hapten-BSA ratio had been reached. This made it possible to prepare, from one hapten in a one-pot reaction, a series of neoglycoconjugates having different, predetermined carbohydrate-carrier ratios. The accuracy of molecular mass determination in SELDI-TOF MS analysis could be increased by using the carrier protein as the internal standard.     

Homogeneous electrogenerated chemiluminescence immunoassay for the determination of digoxin employing Ru(bpy)(2)(dcbpy)NHS and carrier protein.

A highly sensitive homogeneous electrogenerated chemiluminescence (ECL) immunoassay for the determination of anti-digoxin antibody and digoxin hapten was developed employing Ru(bpy)(2)(dcbpy)NHS (bpy = 2,2'-bipyridyl; dcbpy = 2,2'-bipyridine-4,4'-dicarboxylic acid; NHS = N-hydroxysuccinimide ester) as an electrochemiluminescent label and bovine serum albumin (BSA) as a carrier protein. A digoxin hapten was indirectly heavily labelled with Ru(bpy)(2)(dcbpy)NHS through BSA to form Ru(bpy)(2)(dcbpy)NHS-BSA-digoxin conjugate. The ECL intensity of the immunocomplex of the conjugate with anti-digoxin antibody markedly decreased when the immunoreaction between Ru(bpy)(2)(dcbpy)NHS-BSA-digoxin conjugate and anti-digoxin antibody took place. Two formats, direct homogeneous immunoassay for anti-digoxin antibody and competitive immunoassay for digoxin, were developed to determine anti-digoxin antibody and digoxin, respectively. The anti-digoxin antibody concentration in the range 7.6 x 10(-8)-7.6 x 10(-6) g/mL was determined by direct homogeneous format. Digoxin hapten was determined throughout the range 4.0 x 10(-10)-1.0 x 10(-7) g/mL with a detection limit of 1.0 x 10(-10) g/mL by competitive format. The relative standard derivation for 6.0 x 10(-9) g/mL was 4.3%. The method has been applied to assaying digoxin in control human serum.     

Regulation of mouse hepatic ATP-citrate lyase activity by dietary fat evidence for the presence of inactive enzyme

The induction of ATP-citrate lyase activity in mouse liver by dietary carbohydrate (glucose) is markedly reduced by including in the diet a source of polyunsaturated fatty acids. Within 72 h after changing from a standard mouse chow diet to a high carbohydrate diet containing 15% (w/w) of hydrogenated cottonseed oil (as a source of saturated fatty acids), the activity of mouse liver ATP-citrate lyase per milligram cytosolic protein was approx. 3-fold higher than that from mice fed a similar diet containing 15% (w/w) of corn oil. The rate of synthesis of ATP-citrate lyase relative to that for total protein and the rate of degradation of the enzyme were similar for both dietary groups. Elevated levels of enzyme activity in the hydrogenated cottonseed oil-fed livers were not accompanied by a similar increase in the amount of enzyme protein. To explain such findings, we propose that the activity of hepatic ATP-citrate lyase is regulated by dietary polyunsaturated fatty acids through a mechanism involving the conversion of a catalytically active form of the enzyme to a catalytically inactive form. A reversal of this conversion (inactive-active)_is evident within 72 h of removing the mice from the corn oil diet and placing them on the hydrogenated cottonseed oil diet. Futhermore, the conversion appears to be independent of the in vivo rate of synthesis of the enzyme.     

Organomercury bioconjugate synthesis and characterization by matrix-assisted laser desorption ionization mass spectrometry

Synthesis of an organomercury hapten and conjugation of the hapten to proteins and peptides is described. Starting with allylamine, synthesis of the organomercury hapten was completed in five steps using readily available and inexpensive reagents. The key transformation in the synthesis, intramolecular oxymercuration, was achieved in good yield and under mild conditions. Hapten conjugation was afforded via disuccinimide active ester coupling chemistry, and the resulting conjugates were analyzed by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). To exploit the accurate mass measuring capabilities of MALDI-MS, the conjugates were digested with trypsin prior to analysis. The masses of the peptides resulting from tryptic digestion of the organomercury conjugates were accurately measured, and five hapten attachments were identified in the mass range of 1000-2200 m/z. The organomercury bioconjugate synthesized in this study was designed to contain a stable carbon-metal bond, constituting an underutilized approach for preparing protein-metal complexes and may result in mAbs consisting of unique recognition capabilities.     

Artificial peptide and carbohydrate antigens. Immobilization of haptens and adjuvant (MDP) on polyacrylamide

To study the influence of the polyacrylamide carrier on immunogenic properties of the peptide and oligosaccharide haptens, we have prepared artificial antigens by conjugation of a synthetic hexapeptide (homologous to the fragment 95-100 of the murine H-2Db antigen heavy chain) or of an oligosaccharide (antigenic determinant of human blood groops, Lea) with polyacrylamide. In some cases the conjugates containing also a synthetic glycopeptide adjuvant, N-acetylmuramoyl-L-alanyl-D-isoglutamine (MDP), were used. Antisera against haptens were obtained by immunization of BALB/c mice with corresponding conjugates. By the enzyme-linked immunosorbent assay it was shown that these antisera had a high binding titer (up to 10 000) to corresponding hapten, and MDP immobilized on the same carrier as hapten possessed a considerable immunostimulating activity. Thus, usefulness of polyacrylamide for preparation of immunogenic artificial molecules carrying peptide and oligosaccharide haptens was demonstrated.     

Dendrimer versus linear conjugate: Influence of polymeric architecture on the delivery and anticancer effect of paclitaxel

The relative difference in polymeric architectures of dendrimer and linear bis(poly(ethylene glycol)) (PEG) polymer in conjugation with paclitaxel has been described. Paclitaxel, a poorly soluble anticancer drug, was covalently conjugated with PAMAM G4 hydroxyl-terminated dendrimer and bis(PEG) polymer for the potential enhancement of drug solubility and cytotoxicity. Both conjugates were characterized by 1NMR, HPLC, and MALDI/TOF. In addition, molecular conformations of dendrimer, bis(PEG), paclitaxel, and its polymeric conjugates were studied by molecular modeling. Hydrolysis of the ester bond in the conjugate was analyzed by HPLC using esterase hydrolyzing enzyme. In vitro cytotoxicity of dendrimer, bis(PEG), paclitaxel, and polymeric conjugates containing paclitaxel was evaluated using A2780 human ovarian carcinoma cells. Cytotoxicity increased by 10-fold with PAMAM dendrimer-succinic acid-paclitaxel conjugate when compared with free nonconjugated drug. Data obtained indicate that the nanosized dendritic polymer conjugates can be used with good success as anticancer drug carriers.