Site-specific radiolabeling of monoclonal antibodies with biotin/streptavidin

Rosario, Renato B. Del; Wahl, Richard L.

doi:10.1016/0883-2897(89)90067-6

Cited by 8 publications

(5 citation statements)

References 12 publications

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“…However, in a subsequent study using similar reaction conditions, the molecular mass of the mAb-SA conjugate was determined to be about 350 kDa by size-exclusion HPLC (31). SA-mAb conjugates in the 440-600 kDa range also have been reported (32).…”

Section: Discussionmentioning

confidence: 94%

Preparation and Characterization of Anti-Tenascin Monoclonal Antibody−Streptavidin Conjugates for Pretargeting Applications

1999

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Radioimmunopretargeting is based on the separate injection of a modified mAb and the radionuclide and most frequently exploits the very high avidity of biotin for streptavidin (SA). Currently, we are evaluating the therapeutic potential of directly labeled monoclonal antibody (mAb) 81C6, reactive with the extracellular matrix protein tenascin, in surgically created glioma resection cavity patients. To be able to investigate pretargeting in this setting, the synthesis of 81C6 mAb-SA conjugates was required. In the current study, we have evaluated five methods for preparing both murine 81C6 (m81C6) and human/mouse chimeric 81C6 (c81C6) SA conjugates with regard to yield, biotin-binding capacity, immunoreactivity, and molecular weight. The 81C6 mAb and SA were coupled by covalent interaction between sulfhydryl groups generated on the mAb via N-succinimidyl-S-acetylthioacetate, dithiothreitol or 2-iminothiolane (2IT), and maleimido-derivatized SA, prepared via sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) or N-succinimidyl-3-(2-pyridyldithio)-propionate. A noncovalent approach involving reaction of a biotinylated mAb, prepared using biotin caproate, and SA also was studied. The evaluation criteria were yield of mAb-SA 215 kDa monomer, as well as conjugate biotin-binding capacity and immunoreactive fraction. The optimal procedure involved activation of m81C6 or c81C6 with 30 equiv of 2IT and reaction of SA with 10 equiv of SMCC and yielded a conjugate with excellent biotin-binding capacity and immunoreactivity. The ((125)I-labeled m81C6)-2IT-SMCC-SA was stable and did not lose biotin-binding capacity after a 72 h incubation in human glioma cyst fluid in vitro. Although the conjugate was stable in murine serum in vivo, its biotin-binding capacity declined rapidly, consistent with high endogenous biotin levels in the mouse. After injection of the radioiodinated conjugate into athymic mice with subcutaneous D-54 MG human glioma xenografts, high tumor uptake (36.0 +/- 10.7% ID/g at 3 days) and excellent tumor:normal tissue ratios were observed.

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Section: Discussionmentioning

confidence: 94%

Preparation and Characterization of Anti-Tenascin Monoclonal Antibody−Streptavidin Conjugates for Pretargeting Applications

1999

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“…Preliminary experiments indicated that reoxidation of the reduced thiols of 225.28S was competitive (Figure 2, lanes G-N) under the conditions used for the cross-linking reaction. Figure 2 (lanes G-N) shows the nonreducing SDS-PAGE of the resulting product mixtures when samples of 225.28S in which all interchain disulfides have been reduced are chromatographed and allowed to air oxidize and then alkylated with iodoacetamide to block remaining thiols at varying time points (16). The product mixtures consisted of six bands which are assignable (with the migration taken from the top of the gel) to intact H2L2 (molecular weight = 150 000) as well as successive degradations of H2L (=125 000), H2 (=100 000), HL (=75 000), H (=50 000), and L (=25 000) chain fragments resulting from cleavage of interchain disulfide bonds.…”

Section: Resultsmentioning

confidence: 99%

“…The similarity suggests that the molecular mass of the product fragments are approximately 125 000,100 000, and 75 000 and consistent with both H-H and H-L cross-links. The short distance span of ETAC 1 type reagents and the generally accepted hypothesis that heavy and light chains of an IgG molecule remain intimately associated by noncovalent attractions even after cleavage of all interchain disulfides suggests that the reaction products were intramolecularly cross-linked H2L2, H2L, H2, and HL antibody fragments (16,26,27). In this connection, we have also observed (by FPLC and SDS-PAGE) that reduced and iodoacetyl-alkylated heavy and light chains of antibody do not undergo complete dissociation except under strong denaturing conditions at elevated temperatures (16,17).…”

Section: Resultsmentioning

confidence: 99%

“…The recent synthesis of new N-substituted aromatic maleimide derivatives (9,10) and crabescein (15) represent a promising class of organic compounds. We have recently reported our success with the sulfhydryl site-specific labeling of partially reduced monoclonal antibodies with biotin (16,17). An additional caveat in thiol-directed labeling is that alkylation occurs with concomitant and irreversible cleavage of interchain disulfide bonds.…”

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confidence: 99%

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Sulfhydryl site-specific crosslinking and labeling of monoclonal antibodies by a fluorescent equilibrium transfer alkylation crosslink reagent

Rosario¹,

Wahl²,

Brocchini³

et al. 1990

Bioconjugate Chem.

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The site-specific intramolecular cross-linking of sulfhydryls of monoclonal antibodies via a new class of "equilibrium transfer alkylation cross-link (ETAC) reagents" is described. Following complete or partial reduction of interchain disulfides with dithiothreitol (DTT), two murine IgG2a monoclonal antibodies, 225.28S and 5G6.4, were reacted with alpha,alpha-bis[(p-tolylsulfonyl)methyl]-m-aminoacetophenone (ETAC 1a) and a fluorescent conjugated derivative, sulforhodamine B m-(alpha,alpha-bis(p-tolysulfonylmethyl)acetyl)anilide derivative (ETAC 1b). Reducing SDS-polyacrylamide gel electrophoresis analysis of the products from 1b indicated the formation of S-ETAC-S interchain heavy and light chain cross-links (approximately 23-34% overall yield by video-camera densitometry) which do not undergo disulfide-thiol exchange with DTT at 100 degrees C. In contrast, no interchain cross-links were observed upon reaction of unreduced or reduced antibody wherein the thiols have been previously alkylated with iodoacetamide. These results indicated site-specific cross-linking of interchain sulfhydryls and places their distance within 3-4 A. Flow cytometry of the ETAC 1b 5G6.4 cross-linked product using 77 IP3 human ovarian carcinoma target cells showed positive binding and retention of immunoreactivity. The in vivo biodistributions of 131I-labeled intact 5G6.4 and 125I-labeled reduced 5G6.4 + ETAC 1a product in rats were essentially identical over a period of 24 h. The present study illustrates the potential applications of labelable ETAC reagents as thiol-specific probes for a wide variety of immunological studies.

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“…In general, the methods used to achieve agent-antibody conjugation involve the random modification of mAb amino acid side chain amines with drug derivatives and frequently result in an unacceptable reduction in the mAb immunoreactivity at useful levels of drug incorporation (7). Recent approaches have involved the modification of specific sites on the antibody such as the mAb carbohydrate moiety (8) or interchain disulfide bridge (9,10). While use of site-specific labeling techniques avoids many of the pitfalls of random labeling, site-specific labeling methods have the disadvantage of providing immunoconjugates with low levels of agent incorporation.…”

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confidence: 99%

Molecular amplifiers: synthesis and functionalization of a poly(aminopropyl)dextran bearing a uniquely reactive terminus for univalent attachment to biomolecules

Mann¹,

Huang²,

Keana³

1992

Bioconjugate Chem.

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The synthesis and characterization of the versatile dextran-based molecular amplifier 6 is described. Dextran (Mr = 40,200) was selectively monofunctionalized in high yield at its reducing terminus via reductive amination with 2-(4-nitrophenyl)ethylamine to give 1. The nitro group in 1 serves as a masked amino group which is eventually converted into a reactive isothiocyanato group used for monovalent attachment of the completed assembly to a target molecule. Cyanoethylation of 1 gave the terminally nitrophenylated poly(cyanoethyl)dextran 5 which was selectively reduced to the corresponding poly(aminopropyl) derivative 6 with BH3.THF, a reagent which preserved the end nitro group. Conjugation of amplifier 6 with the isothiocyanate-derivatized Gd(III) chelate 7 gave conjugate 9 containing about 22 mol of chelate/mol of amplifier. The T1 relaxivity per Gd(III) ion of 9 in H2O was 15.0 mM-1 s-1, about 3-fold higher than that of free Gd(III)DTPA in H2O. The nitro group of 9 was then selectively reduced to the corresponding amine 10, which was converted into isothiocyanate 11. The reactivity of the single isothiocyanate group in 11 was demonstrated by coupling to 5-aminoeosin, giving conjugate 12. Amplifier 6 was also conjugated with the acid-labile N-cis-aconityl derivative 8 of the potent anticancer agent daunomycin. The nitro group of the resulting conjugate 13 was then reduced and the resulting amine 14 was converted into mono isothiocyanate 15. Compound 15 reacted with a water-insoluble amine-containing solid support to give 16. Free daunomycin was released from 16 by exposure to citrate-phosphate buffer at pH 4.0.

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Site-specific radiolabeling of monoclonal antibodies with biotin/streptavidin

Cited by 8 publications

References 12 publications

Preparation and Characterization of Anti-Tenascin Monoclonal Antibody−Streptavidin Conjugates for Pretargeting Applications

Preparation and Characterization of Anti-Tenascin Monoclonal Antibody−Streptavidin Conjugates for Pretargeting Applications

Sulfhydryl site-specific crosslinking and labeling of monoclonal antibodies by a fluorescent equilibrium transfer alkylation crosslink reagent

Molecular amplifiers: synthesis and functionalization of a poly(aminopropyl)dextran bearing a uniquely reactive terminus for univalent attachment to biomolecules

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