Biotin reagents for antibody pretargeting. 5. Additional studies of biotin conjugate design to provide biotinidase stability.

An investigation was conducted in which the stabilities of four structurally different biotin derivatives were assessed with regard to biotinamide bond hydrolysis by the enzyme biotinidase. The biotin derivatives studied contained an extra methylene in the valeric acid chain of biotin (i.e., homobiotin), or contained conjugated amino acids having hydroxymethylene, carboxylate, or acetate functionalities on a methylene alpha to the biotinamide bond. The biotinidase hydrolysis assay was conducted on biotin derivatives that were radioiodinated at high specific activity, and then subjected to diluted human serum at 37 degrees C for 2 h. After incubation, assessment of biotinamide bond hydrolysis by biotinidase was readily achieved by measuring the percentage of radioactivity that did not bind with avidin. As controls, an unsubstituted biotin derivative which is rapidly cleaved by biotinidase and an N-methyl-substituted biotin derivative which is stable to biotinidase cleavage were included in the study. The results indicate that increasing the distance from the biotin ring structure to the biotinamide bond by one methylene only decreases the rate of biotinidase cleavage, but does not block it. The data obtained also indicate that placing a hydroxymethylene, carboxylate, or acetate alpha to the biotinamide bond is effective in blocking the biotinamide hydrolysis reaction. These data, in combination with data previously obtained, which indicate that biotin derivatives containing hydroxymethylene or carboxylate moieties retain the slow dissociation rate of biotin from avidin and streptavidin [Wilbur, D. S., et al. (2000) Bioconjugate Chem. 11, 569-583], strongly support incorporation of these structural features into biotin derivatives being used for in vivo targeting applications.     

Evaluation of biotin-dye conjugates for use in an HPLC assay to assess relative binding of biotin derivatives with avidin and streptavidin

In this investigation, studies were conducted to determine if size exclusion HPLC could be used to assess relative association rates (on-rates) and dissociation rates (off-rates) of biotin derivatives from avidin (Av) and streptavidin (SAv). For easy detection and quantification of biotin derivatives, molecules that can be detected by UV absorbance were conjugated to biotin. Concern that conjugation of the chromophoric moieties (dyes) might affect biotin binding with Av and SAv or might interact with the HPLC column led to evaluation of 10 biotin-dye conjugates. The dyes conjugated with biotin included dansyl, cyanocobalamin (CN-Cbl), coumarin 343, Lissamine-rhodamine, fluorescein, Cascade Blue, Lucifer Yellow, Oregon Green, tetramethylrhodamine, and Alexa Fluor 594. The biotin-dye conjugates were initially evaluated to determine their peak characteristics on two different size exclusion HPLC columns. Measurement of the percent of biotin-dye conjugate bound with Av in the presence of an equal quantity of biotin provided an association rate relative to biotin. All of the biotin-dyes tested had association rates within a factor of 3x (slower) that of biotin. The relative dissociation rate of biotin-dye conjugates was assessed by challenging the biotin conjugate bound to Av or SAv with a large excess of biotin. All of the initial biotin-dye conjugates tested bound Av and SAv tightly resulting in very slow dissociation rates. From the biotin-dye conjugates studied, biotin-CN-Cbl, 6b, was selected as the best conjugate for the HPLC assay. To test the HPLC assay, an iminobiotin-CN-Cbl conjugate, 13a, and a biotin-sarcosine-CN-Cbl conjugate, 13b, were synthesized. The fact that the iminobiotin does not bind with Av at physiological pH was easily detected in the size exclusion HPLC assay. The biotin-sarcosine-CN-Cbl conjugate was expected to have a more rapid dissociation rate than the other biotin-dye conjugates. This was confirmed in that HPLC assay. Although 13b bound tightly with Av in the absence of added biotin, it was completely released within 1 h when challenged by an excess of biotin. A slower dissociation of 13b was noted with SAv. The results obtained indicate that CN-Cbl conjugates of biotin derivatives can be used to determine relative on-rates and off-rates of biotin derivatives with Av and SAv. The studies also demonstrated that the biotin-CN-Cbl conjugate, 6b, can be used as a reference compound to compare on-rates and off-rates of nonchromophoric biotin derivatives.     

Biotin reagents for antibody pretargeting. 7. Investigation of chemically inert biotinidase blocking functionalities for synthetic utility

An investigation was conducted to evaluate three biotin derivatives designed to block biotinidase cleavage of the biotinamide bond. Difficulties in multistep syntheses of molecules containing tert-butyl protected hydroxymethyl and carboxylate groups positioned alpha to a biotinamide bond led to the investigation of alternative biotinidase-blocking moieties that do not require protection and deprotection. The targeted biotin derivatives contained serine-O-methyl ether, 2-aminobutyric acid, and valine moieties conjugated to the biotin carboxylate functionality. Those derivatives were further modified with a radioiodinated aryl ring to study their biotinidase stability. As a comparison to previously studied biotin derivatives, radioiodinated versions of biotin conjugates that contained (a) no biotinidase stabilizing group, (b) an N-methyl (sarcosine) stabilizing group, (c) an alpha-carboxylate (aspartate) stabilizing group and hydroxymethyl (serine) stabilizing group were also prepared and tested. When tested in human serum, all of the radioiodinated biotinidase-stabilized biotin derivatives had <1% biotinamide cleavage. Thus, under the conditions studied, all of the tested biotinidase blocking moieties appeared to be equal with regards to protection from biotinidase cleavage. Further testing of the biotin derivatives included a HPLC assay to determine their relative dissociation from recombinant streptavidin (rSAv). The dissociation of cyanocobalamin (CN-Cbl) adducts of biotin-serine-O-methyl ether, biotin-aminobutyric acid, and biotin-valine were compared with the CN-Cbl adduct of biotin-sarcosine. The relative rates of dissociation found were biotin-sarcosine-CN-Cbl > biotin-valine-CN-Cbl > biotin-serine-O-methyl ether-CN-Cbl > biotin-aminobutyric acid-CN-Cbl. Due to the high cost of serine-O-ethyl ether (and its N-Boc derivative) and difficulty in syntheses of its biotin derivatives, that adduct is not an attractive candidate for application to compounds used in vivo. The higher lipophilicity and diminished binding of the biotin-valine adduct also makes its use in vivo less attractive. Thus, the biotin-aminobutyric acid adduct appears to be the best candidate for incorporation into biotin derivatives used in vivo, as it simplifies the synthetic procedures, has low cost, and provides effective blocking of biotinidase while retaining high binding affinity.     

A biotin-protein bond with stability in plasma

A nonradioactive label for peptide hormones would be useful for pharmacokinetic studies in infants, children, and pregnant women. Because the binding affinity between biotin and avidin is large (Ka=10(15) M(-1)), biotin could also serve as a covalent label for subsequent detection using a variety of avidin conjugates. However, biotin labels produced by most commercially available biotinylating reagents are rapidly cleaved from protein in plasma. We sought to synthesize a stable biotin label for protein. With the use of immunoglobulin G (IgG) as a model protein, biotin was conjugated through a cysteine residue; a carboxylate group was positioned alpha to the biotinamide bond. Stability of the bond in the presence of plasma and buffer control was assessed by release of biotin. Released biotin was separated from biotinylated IgG by ultrafiltration and was quantitated by an avidin-binding assay. In plasma, less than 0.6% of bound biotin was released. This release rate is not significantly different from buffer and is less than 7% of the release rate for IgG biotinylated by N-hydroxysuccinimide-LC-biotin. We conclude that this biotin-protein bond is stable in plasma. We speculate that many uses of avidin-biotin technology could be improved by using this method for protein labeling.     

Biotin-4-fluorescein based fluorescence quenching assay for determination of biotin binding capacity of streptavidin conjugated quantum dots

The valency of quantum dot nanoparticles conjugated with biomolecules is closely related to their performance in cell tagging, tracking, and imaging experiments. Commercially available streptavidin conjugates (SAv QDs) are the most commonly used tool for preparing QD-biomolecule conjugates. The fluorescence quenching of biotin-4-fluorscein (B4F) provides a straightforward assay to quantify the number of biotin binding sites per SAv QD. The utility of this method was demonstrated by quantitatively characterizing the biotin binding capacity of commercially available amphiphilic poly(acrylic acid) Qdot ITK SAv conjugates and poly(ethylene glycol) modified Qdot PEG SAv conjugates with emission wavelengths of 525, 545, 565, 585, 605, 625, 655, 705, and 800 nm. Results showed that 5- to 30-fold more biotin binding sites are available on ITK SAv QDs compared to PEG SAv QDs of the same color with no systematic variation of biotin binding capacity with size.     

Polymer-bound reagents for the introduction of spacer-modified biotin labels

We have developed a method for the chemoselective introduction of spacer modified biotin labels into unprotected multi-functional amines. A range of novel biotin spacer conjugates attached to a polymer-bound sulfonamide anchor was prepared using established amide bond forming procedures. After chemical transformation of the attachment site by alkylation, the resulting reactive species were utilized as N-selective polymer-supported biotinylation reagents. The labeled compounds, obtained in good to excellent yield and purity, are free of residual biotin and possess a custom tailored distance from the immobilization site being especially suited for the immobilization on streptavidin-functionalized dextran layers of surface plasmon resonance detector chips. In addition, derivatives displaying a phenyl group were synthesized in order to demonstrate the versatility of the procedure for the simultaneous introduction of spacer-modified biotin and a UV-light absorbing moiety. The formation of biotin sulfoxides in the presence of in situ generated peroxides was investigated and is discussed. Our results suggest that this derivatization technique is a useful addition to the existing biotin labeling protocols.     

Facile synthesis of multivalent nitrilotriacetic acid (NTA) and NTA conjugates for analytical and drug delivery applications

High-affinity nitrilotriacetic acids (NTA) have great potential in the molecular manipulation of His-tagged proteins. We have developed a facile method to synthesize multivalent NTA and its conjugates. Starting with appropriately protected lysine, we synthesized the mono-NTA synthons functionalized with either an amino group or a carboxylic group. We then obtained tri-NTA through the condensation of the amino NTA and the carboxylic NTA. Using amino tri-NTA as the key intermediate, we synthesized a series of tri-NTA conjugates with a variety of functional units including biotin, dialkyl, fluorescein, and a hydroxybenzimidate moiety. The biotin-tri-NTA was employed to convert a Biacore streptavidin chip into a high-affinity tri-NTA chip. The equilibrium dissociation constants of tri-NTA/His-tagged protein complexes measured by surface plasmon resonance are in the 20 nM range. Histidine(6)-tagged yeast cytosine deaminase (His6-yCD) was incorporated onto the liposome surface by the lipid-tri-NTA conjugate without any activity loss. Fluorescein-tri-NTA formed a stable 1:1 complex with His6-yCD without significant fluorescence quenching. Specific tri-NTA derivatives for the radiolabeling and coupling of two His-tagged proteins to each other are described. Thus, we have added to the toolbox a number of high-affinity tri-NTA adaptors for the manipulation of His-tagged molecules.     

Chemically synthesized non-radioactive biotinylated long-chain nucleic acid hybridization probes.

A new method for the chemical labelling of nucleic acid with biotin to produce non-radioactive probes has been developed. NN'-Bis-(3-aminopropyl)butane-1,4-diamine (spermine) and long-chain diamino compounds (diaminohexane, diaminodecane and diaminododecane) were linked covalently to biotin and the resultant conjugates were attached to nucleic acid by using a cross-linking reagent (glutaraldehyde or diepoxyoctane). Iodoacetylation and biotinylation of the long-chain diamino compounds produced modified biotinylated conjugates that can be linked to DNA without the use of a cross-linking reagent. These types of probes attach one biotin molecule to each linker arm of spermine, diamino and iodoacetylated amino derivatives. Such probes have long linker arms separating the biotin moiety from the hybridization sites of the nucleic acid. These probes can detect 10 pg of target DNA by dot-blot hybridization.     

A universal approach to the synthesis of carbohydrate conjugates of polyhedral boron compounds as potential agents for boron neutron capture therapy

A universal approach to the synthesis of carbohydrate conjugates with polyhedral boron compounds (PBCs) was developed. Oligosaccharide derivatives with amino group in aglycone moiety can be conjugated with PBC carboxy derivatives using N-methyl-N-(4,6-dimethoxy-1,3,5-triazin-2-yl)morpholinium chloride as a condensing agent. Both N-and O-glycosides differing in the conformation mobility around the glycoside bond were shown to be useful as oligosaccharides with a functional group in the aglycone moiety. This allows the application of this approach to the synthesis of PBC conjugates with a wide range of oligosaccharides isolated from natural sources can be transformed into N-glycosides with a functional group in aglycone. The approach was tested by conjugation of the carboxy derivatives of ortho-carborane and dodecaborate anion with lactose as a model oligosaccharide. Lactose, an easily available disaccharide, is a ligand of lectins expressed on the surface of melanoma cells. The approach suggested is the first example of the synthesis of such conjugates that does not require protective groups for the carbohydrate residue. It is especially important for obtaining dodecaborate-carbohydrate conjugates for which the removal of protective groups is often a non-trivial task.     

Biotin reagents in antibody pretargeting. 6. Synthesis and in vivo evaluation of astatinated and radioiodinated aryl- and nido-carboranyl-biotin derivatives

An investigation has been conducted to prepare and evaluate several radiohalogenated biotin derivatives as part of our studies to develop reagents for carrying (211)At in cancer pretargeting protocols. The primary goal of the investigation was to determine the in vivo stability and distribution properties of astatinated biotin derivatives. In addition to astatination, the biotin derivatives were radioiodinated for in vitro and in vivo comparison. Biodistributions were conducted in athymic mice, with sacrifice times of 1, 4, and 24 h to correspond to 9%, 32%, and 90% of (211)At decay (t(1/2) = 7.21 h). In the investigation, two biotin derivatives, 1a and 2a, were synthesized which had structures that contain a biotin moiety, a biotinidase-blocking moiety, an ether linker moiety, and an aryl stannane moiety for radiohalogenation. Biotin derivatives 1a and 2a were radiolabeled with (125/131)I to give [(125)/(131)I]1b or [(125)I]2b and with (211)At to give [(211)At]1c or [(211)At]2c. In vivo studies demonstrated that co-injected [(125)I]2b and [(131)I]1b had very similar tissue distributions in athymic mice. Co-injection of [(211)At]2c and [(125)I]2b provided data that indicated that rapid deastatination occurred in vivo. A second set of biotin derivatives, 3a, 4a, and 5a, were synthesized which had structures that contain a biotin moiety, a biotinidase-blocking moiety, and an anionic nido-carborane moiety for radiohalogenation. The biotin derivatives 4a and 5a contained an aryl moiety not present in 3a, and 5a had a trialkylamine functionality not present in 3a or 4a. Biotin derivative 3a was radioiodinated, but was not further investigated. Biotin derivatives 4a and 5a were radiolabeled with (211)At and (125)I to produce [(125)I]4b/[(211)At]4c and [(125)I]5b/[(211)At]5c. Comparison of [(125)I]4b and (separately) [(125)I]5b with [(131)I]1b showed that the nido-carborane containing biotin derivatives were retained in blood and tissue more than the aryl iodide derivative. In vivo evaluations of [(211)At]4c/[(125)I]4b and (separately) [(211)At]5c/[(125)I]5b indicated that some deastatination occurred in these compounds, but it was much less than observed for the aryl derivative [(211)At]2c. While the nido-carborane containing biotin derivatives provide a significant improvement in astatine stability over biotin derivatives previously studied, additional derivatives need to be prepared and studied to further improve the in vivo stability and blood/tissue clearance of these compounds.     

A uniform approach to the synthesis of carbohydrate conjugates of polyhedral boron compounds as potential agents for boron neutron capture therapy

A uniform approach to the synthesis of carbohydrate conjugates with polyhedral boron compounds (PBCs) was developed. Oligosaccharide derivatives with an aglycone moiety amino group can be coupled with PBC carboxyl derivatives using N-methyl-N-(4,6-dimethoxy-1,3,5-triazin-2-yl)morpholinium chloride as a coupling agent. Both N- and O-glycosides differing in the conformational mobility around the glycoside bond were shown to be useful as oligosaccharides with a functional group in the aglycone moiety. This allows the application of this approach to the synthesis of PBC conjugates with a wide range of oligosaccharides. For example, not only oligosaccharides obtained by chemical synthesis but also reducing oligosaccharides isolated from natural sources can be transformed into N-glycosides. The approach was tested by the example of conjugation of the carboxyl derivatives of ortho-carborane and dodecaborate anion with lactose as a model oligosaccharide. Lactose, an easily available disaccharide, is a ligand for lectins expressed on the surface of melanoma cells. The approach suggested is the first example of the synthesis of such conjugates that does not require protective groups for the carbohydrate residue. It is especially important for obtaining dodecaborate-carbohydrate conjugates for which the removal of protective groups is often a non-trivial task.     

Reagents for astatination of biomolecules. 2. Conjugation of anionic boron cage pendant groups to a protein provides a method for direct labeling that is stable to in vivo deastatination

Cancer-targeting biomolecules labeled with 211At must be stable to in vivo deastatination, as control of the 211At distribution is critical due to the highly toxic nature of alpha-particle emission. Unfortunately, no astatinated aryl conjugates have shown in vivo stability toward deastatination when (relatively) rapidly metabolized proteins, such as monoclonal antibody Fab' fragments, are labeled. As a means of increasing the in vivo stability of 211At-labeled proteins, we have been investigating antibody conjugates of boron cage moieties. In this investigation, protein-reactive derivatives containing a nido-carborane (2), a bis-nido-carborane derivative (Venus Flytrap Complex, 3), and four 2-nonahydro-closo-decaborate(2-) derivatives (4-7) were prepared and conjugated with an antibody Fab' fragment such that subsequent astatination and in vivo tissue distributions could be obtained. To aid in determination of stability toward in vivo deastatination, the Fab'-borane conjugates were also labeled with 125I, and that material was coinjected with the 211At-labeled Fab'. For comparison, direct labeling of the Fab' with 125I and 211At was conducted. Direct labeling with Na[125I]I and Chloramine-T gave an 89% radiochemical yield. However, direct labeling of the Fab' with Na[211At]At and Chloramine-T resulted in a yield of <1% after quenching with NaS2O5. As another comparison, the same Fab' was conjugated with p-[211At]astatobenzoate NHS ester, [211At]1c-Fab', and (separately) with p-[125I]iodobenzoate NHS ester, [125I]1b-Fab'. An evaluation in athymic mice demonstrated that [211At]1c-Fab' underwent deastatination. In contrast, the high in vivo stability of [125I]1b-Fab' allowed it to be used as a tracer control for the natural distribution of Fab'. Although found to be much more stable in vivo than [211At]1c-Fab', the biodistributions of nido-carborane conjugated Fab' ([125I]2-Fab'/ [211At]2-Fab') and the bis-nido-carborane (VFC) ([125I]3-Fab'/[211At]3-Fab') had very different in vivo distributions than the control [125I]1b-Fab'. Biodistributions of closo-decaborate(2-) conjugates ([125I]4-Fab'/[211At]4-Fab', [125I]6-Fab'/[211At]6-Fab', and [125I]7-Fab'/[211At]7-Fab') demonstrated that they were stable to in vivo deastatination and had distributions similar to that of the control [125I]1b-Fab'. In contrast, a benzyl-modified closo-decaborate(2-) derivative evaluated in vivo ([125I]5-Fab'/[211At]5-Fab') had a very different tissue distribution from the control. This study has shown that astatinated protein conjugates of closo-decaborate(2-) are quite stable to in vivo deastatination and that some derivatives have little effect on the distribution of Fab'. Additionally, direct 211At labeling of Fab' conjugated with closo-decaborate(2-) derivatives provide very high (e.g., 58-75%) radiochemical yields. However, in vivo data also indicate that the closo-decaborate(2-) may cause some retention of radioactivity in the liver. Studies to optimize the closo-decaborate(2-) conjugates for protein labeling are underway.     

Metabolic biotinylation of cell surface receptors for in vivo imaging

We have developed a versatile, potent technique for imaging cells in culture and in vivo by expressing a metabolically biotinylated cell-surface receptor and visualizing it with labeled streptavidin moieties. The recombinant reporter protein, which incorporates a biotin acceptor peptide (BAP) between an N-terminal signal sequence and a transmembrane domain, (BAP-TM) was efficiently biotinylated by endogenous biotin ligase in mammalian cells with the biotin displayed on the cell surface. Tumors expressing the BAP-TM have high sensitivity for magnetic resonance and fluorescence tomographic imaging in vivo after intravascular injection of streptavidin conjugated to magnetic nanoparticles or fluorochromes, respectively. Moreover, streptavidin-horseradish peroxidase conjugates in conjunction with a peroxidase-sensitive gadolinium agent further increased and prolonged the magnetic resonance signal. This BAP-TM allows noninvasive real-time imaging of any cell type transduced to express this reporter protein in culture or in vivo.     

Investigations of the MceIJ-catalyzed posttranslational modification of the microcin E492 C-terminus: linkage of ribosomal and nonribosomal peptides to form "trojan horse" antibiotics

MceIJ is a two protein complex responsible for attachment of a C-glycosylated and linearized derivative of enterobactin, an iron scavenger (siderophore) and product of nonribosomal peptide synthetase machinery, to the C-terminal serine residue of microcin E492 (MccE492), an 84 aa ribosomal antibiotic peptide produced by Klebsiella pneumoniae RYC492. The MceIJ-catalyzed formation of the glycosyl ester linkage between MccE492 and the siderophore requires ATP and Mg(II) as cofactors. This work addresses the ATP utilization, mechanism of C-terminal carboxylate activation, and substrate scope of MceIJ. Formation of the ribosomal peptide-nonribosomal peptide linkage between the MccE492 C-terminal decapeptide and monoglycosylated enterobactin (MGE) requires cleavage of the alpha,beta bond of ATP and formation of a putative peptidyl-CO-AMP intermediate. Attack of the peptidyl-CO-AMP carbonyl by the deprotonated C4' hydroxyl of the glucose moiety forms a glycosyl ester linkage with release of AMP. Site-directed mutagenesis of the three cysteines and five histidines in MceI to alanines reveals that these residues are not required structurally or catalytically. MceIJ recognizes all glycosylated enterobactin derivatives formed by the MccE492 gene cluster members MceC ( C-glycosyltransferase) and MceD (esterase) in vitro and a MGE derivative lacking the C6' hydroxyl moiety. The protein complex also accepts and modifies the C-terminal decapeptide substrate fragments of the structurally related microcins H47, I47, and M. MccE492 C-terminal decapeptides bearing fluorescein and biotin moieties on the N-terminus are also substrates for MceIJ, which provides a route for the chemoenzymatic synthesis of enterobactin conjugates with peptide linkages.     

Amino acids/peptides conjugated heterocycles: A tool for the recent development of novel therapeutic agents

Amino acids/peptide conjugated heterocycles represent an important class of therapeutical agents. Biologically active heterocycles are conjugated with amino acids or peptides to increase the drug resistance. Furthermore, the amino acid/peptide based drugs have low toxicity, ample bioavailability and permeability, modest potency and good metabolic and pharmacokinetic properties. Synthetic amino acid/peptides based heterocyclic conjugates constitute a promising choice for the development of new, less toxic and safer conventional pharmaceutical drugs in the near future. In this review, we discuss and highlight the recent findings of the structural features that encourage biological applications of amino acid/peptides based conjugates.     

A human biotin acceptor domain allows site-specific conjugation of an enzyme to an antibody-avidin fusion protein for targeted drug delivery

We have previously constructed an antibody-avidin (Av) fusion protein, anti-transferrin receptor (TfR) IgG3-Av, which can deliver biotinylated molecules to cells expressing the TfR. We now describe the use of the fusion protein for antibody-directed enzyme prodrug therapy (ADEPT). The 67 amino acid carboxyl-terminal domain (P67) of human propionyl-CoA carboxylase alpha subunit can be metabolically biotinylated at a fixed lysine residue. We genetically fused P67 to the carboxyl terminus of the yeast enzyme FCU1, a derivative of cytosine deaminase that can convert the non-toxic prodrug 5-fluorocytosine to the cytotoxic agent 5-fluorouracil. When produced in Escherichia coli cells overexpressing a biotin protein ligase, the FCU1-P67 fusion protein was efficiently mono-biotinylated. In the presence of 5-fluorocytosine, the biotinylated fusion protein conjugated to anti-rat TfR IgG3-Av efficiently killed rat Y3-Ag1.2.3 myeloma cells in vitro, while the same protein conjugated to an irrelevant (anti-dansyl) antibody fused to Av showed no cytotoxic effect. Efficient tumor cell killing was also observed when E. coli purine nucleoside phosphorylase was similarly targeted to the tumor cells in the presence of the prodrug 2-fluoro-2'-deoxyadenosine. These results suggest that when combined with P67-based biotinylation, anti-TfR IgG3-Av could serve as a universal delivery vector for targeted chemotherapy of cancer.     

Trifunctional conjugation reagents. Reagents that contain a biotin and a radiometal chelation moiety for application to extracorporeal affinity adsorption of radiolabeled antibodies

A method of removing radiolabeled monoclonal antibodies (mAbs) from blood using a device external to the body, termed extracorporeal affinity-adsorption (EAA), is being evaluated as a means of decreasing irradiation of noncancerous tissues in therapy protocols. The EAA device uses an avidin column to capture biotinylated-radiolabeled mAbs from circulated blood. In this investigation, three trifunctional reagents have been developed to minimize the potential deleterious effect on antigen binding brought about by the combination of radiolabeling and biotinylation of mAbs required in the EAA approach. The studies focused on radiolabeling with (111)In and (90)Y, so the chelates CHX-A' '-DTPA and DOTA, which form stable attachments to these radionuclides, were incorporated in the trifunctional reagents. The first trifunctional reagent prepared did not incorporate a group to block the biotin cleaving enzyme biotinidase, but the two subsequent reagents coupled aspartic acid to the biotin carboxylate for that purpose. All three reagents used 4,7,10-trioxa-1,13-tridecanediamine as water-soluble spacers between an aminoisophthalate core and the biotin or chelation group. The mAb conjugates were radioiodinated to evaluate cell binding as a function of substitution. Radioiodination was used so that a direct comparison with unmodified mAb could be made. Evaluation of the number of conjugates per antibody versus cell binding immunoreactivities indicated that minimizing the number of conjugates was best. Interestingly, a decrease of radioiodination yield as a function of the number of isothiocyanate containing conjugates per mAb was noted. The decreased yields were presumably due to the presence of thiourea functionality formed in the conjugation reaction. Radiolabeling with (111)In and (90)Y was facile at room temperature for conjugates containing the CHX-A' ', but elevated temperature (e.g., 45 degrees C) was required to obtain good yields with the DOTA chelate. Stability of (90)Y labeled mAb in serum, and when challenged with 10 mM EDTA, was high. However, challenging the (90)Y labeled mAb with 10 mM DTPA demonstrated high stability for the DOTA containing conjugate, but low stability for the CHX-A' ' containing conjugate. Thus, the choice between these two chelating moieties might be made on requirements for facile and gentle labeling versus very high in vivo stability. Application of the trifunctional biotinylation reagents to the blood clearance of labeled antibodies in EAA is under investigation. The new reagents may also be useful for other applications.     

The synthesis of polyamide-oligonucleotide conjugate molecules.

We have developed methods for the synthesis of peptide-oligodeoxyribonucleotide conjugate molecules in particular, and polyamide-oligonucleotide conjugates in general. Synthesis is carried out by a solid-phase procedure and involves the assembly of a polyamide on the solid support, conversion of the terminal amino group to a protected primary aliphatic hydroxy group by reaction with alpha, omega-hydroxycarboxylic acid derivatives, and finally oligonucleotide synthesis using phosphoramidite chemistry. The conjugate molecules can be used as DNA probes, with the polyamide component carrying one or more non-radioactive markers. These conjugates also have the potential to be used as anti-sense inhibitors of gene expression, with the peptide segment acting as a targeting moiety.     

Novel chloroenyne-modified amino acid derivatives

Three groups of chloroenyne-modified amino acids were synthesized. Chloroenyne moiety was attached at the N- or C-terminal amino acid (Tyr, Phe, Val, Gly, Lys) position carrying different protecting groups. Prepared derivatives will be used as building blocks in the synthesis of enediyne-peptide conjugates. Furthermore, reactivity of modified amino acids in the peptide bond formation reaction was tested.     

Synthesis of poly(ethylene glycol)-based saquinavir prodrug conjugates and assessment of release and anti-HIV-1 bioactivity using a novel protease inhibition assay

Various poly(ethylene glycol)(PEG)-based prodrug conjugates of the HIV-1 protease inhibitor (PI) saquinavir (SQV) were prepared using several types of chemical groups potentially capable of modifying its pharmacokinetic properties. These prodrug conjugates included SQV-cysteine-PEG3400, SQV-cysteine-PEG3400-biotin, SQV-cysteine(R.I.CK-Tat9) [a cationic retro-inverso-cysteine-lysine-Tat nonapeptide]-PEG3400, and SQV-cysteine(R.I.CK(stearate)-Tat9)-PEG3400. SQV was linked to cysteine to form a releasable SQV-cysteine ester bond in all of the conjugates. The amino group of the cysteine moiety provided an attachment site for a slower-degrading amide bond with N-hydroxysuccinimide-activated forms of PEG- and PEG-biotin. Disulfide bonds were used to attach the cationic peptides, R.I.CK-Tat9 and R.I.CK(stearate)-Tat9 to the cysteine moiety in order to provide cell-specific release. An assay was established and validated for measuring the activity of SQV and other protease inhibitors in biological samples. In this assay, cleavage of an internally quenched fluorescent substrate, Arg-Glu(EDANS)-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gly-Lys(DABCYL)-Arg by HIV-1 protease was inhibited by SQV in a dose-dependent manner at concentrations of 0.05-0.5 microM. All prodrug conjugates were shown to be inactive in this assay until the ester bond was cleaved and active SQV was released. The prodrug reconversion half-lives in 0.1 N HCl, phosphate-buffered saline (PBS) at pH 7.4 and in spiked plasma at 37 degrees C were 9, 14, and 0.9 h, respectively. The anti-HIV-1 activity (ED(50)) of the PEG-based SQV prodrug conjugates was evaluated in MT-2 cells using an MTT assay. The activity of conjugated SQV was reduced (ED(50) = 900 nM) for the PEG only conjugate, but restored with the addition of biotin (ED(50) = 125 nM), R.I.CK-Tat9 (ED(50) = 15 nM), and R.I.CK(stearate)-Tat9 (ED(50) = 62 nM) as compared to maximum achievable anti-HIV-1 activity (unconjugated SQV, control, ED(50) = 15 nM), suggesting enhanced cellular uptake of conjugates. Cytotoxicity (LD(50)) was assessed for all prodrug conjugates using non-HIV-1 infected cells and was found to be in the micromolar range. The difference between the LD(50) and ED(50) suggests a favorable therapeutic index for the prodrug conjugates. In conclusion, these promising initial results demonstrate that the reconversion of the conjugate prodrugs was complete and that active SQV was released. Since the major delivery advantages of PEG prodrug conjugates can only be observed in vivo, issues of reconversion and elimination half-lives in plasma will have to be further studied in an in vivo model. The current results also demonstrate that the protease inhibition assay is a simple yet effective bioanalytical tool that can be used to assess the release and anti-HIV-1 activity of HIV-1 PIs from their prodrug forms.