Conjugation of antibodies with bifunctional chelating agents: Isothiocyanate and bromoacetamide reagents, methods of analysis, and subsequent addition of metal ions

Meares, Claude F.; McCall, Michael; Reardan, Dayton T.; Goodwin, David A.; Diamanti, Carol I.; McTigue, Maureen

doi:10.1016/0003-2697(84)90517-7

Cited by 335 publications

(224 citation statements)

References 21 publications

Supporting

Mentioning

221

Contrasting

Unclassified

Order By: Relevance

“…The lysine e-NH 2 group has been widely used to link chemical reagents to peptides (Meares et al, 1984;Terrillon et al, 2002). We used a slight molar excess of DOTA to NHS/DCC in order to activate only one carboxyl group and prevent DOTA from bridging two or more dLVT molecules.…”

Section: Discussionmentioning

confidence: 99%

Improved radiotracing of oxytocin receptor-expressing tumours using the new [111In]-DOTA-Lys8-deamino-vasotocin analogue

et al. 2003

View full text Add to dashboard Cite

Oxytocin receptors (OTR) have been described in a number of tumours of different origin, and represent a new target for specific radiolabelled oxytocin (OT) analogues in cancer diagnosis and therapy. By linking the DOTA chelating agent to position 8 of the deamino derivative of Lys 8 -vasotocin (dLVT), we obtained a new compound (DOTA-dLVT) with the following characteristics: (1) it forms a monomeric and stable compound that binds to OTR with an affinity comparable to that of the endogenous OT ligand; (2) it is characterised by a very good selectivity profile for the human OTR, with a low affinity binding to the closely related V1a, V1b and V2 vasopressin receptor subtypes; (3) it induces rapid and persistent receptor internalisation and (4) when radiolabelled, [ 111 In]-DOTA-dLVT is efficiently and selectively taken up by OTR-positive tumours grown in mice. These features makes radiolabelled DOTA-dLVT a very good candidate for the radiotargeting of OTR-expressing tumours.

show abstract

Section: Discussionmentioning

confidence: 99%

Improved radiotracing of oxytocin receptor-expressing tumours using the new [111In]-DOTA-Lys8-deamino-vasotocin analogue

et al. 2003

View full text Add to dashboard Cite

show abstract

“…Following reduction, two protein conjugation reactions were prepared as follows: a small scale analytical conjugation that used the trace-labeled chelator and a large scale conjugation that used unlabeled chelator. The small scale conjugation was used to monitor the extent of the conjugation reaction (32).…”

Section: Methodsmentioning

confidence: 99%

Site-specific Conjugation on Serine → Cysteine Variant Monoclonal Antibodies

Stimmel¹,

Merrill²,

Kuyper³

et al. 2000

Journal of Biological Chemistry

View full text Add to dashboard Cite

We have engineered a cysteine residue at position 442 (EU/OU numbering) in the third constant domain (C H 3) of the heavy chain of several IgGs with different specificities, isoforms, and variants with the intent to introduce a site for chemical conjugation. The variants were expressed in NS0 mouse myeloma cells, where monomeric IgG is the major form and formation of aggregate was minimal. Monomeric IgG contained no free thiol; however, it was discovered that the engineered thiols were reversibly blocked and could be reduced under controlled conditions. Following reduction, reactive thiol was conjugated with a cysteine-specific bifunctional chelator, bromoacetyl-TMT to a humanized 323/A3 IgG4 variant. Conjugation had no significant effect on antibody affinity. To prove that the conjugation was site-specific, an antibody-TMT conjugate was labeled with lutetium-177 and subjected to peptide mapping followed by sequence analysis. Glu-C digestion demonstrated that 91% of the label was recovered in the COOH-terminal peptide fragment containing the engineered cysteine.

show abstract

“…This was corroborated by an instant thin-layer chromatography (ITLC) assay (Meares et al, 1984), in which aliquots of the radiolabelled immunoconjugates were spotted in duplicate onto preactivated (heated to 1 I0C for 30 min) mILC plates (Gelman Sciene), and developed with 0.1 M sodium citrate buffer pH 5.0. The strips were cut into four pieces and counted using an LKB gamma counter.…”

Section: Materis and Methodsmentioning

confidence: 99%

“…This treatment typically introduced 1 thiol per chimeric B72.3. A malimide derivative of the chelator [9N3 macrocycle (CT82, Figure 1), CT-DTPA (CT74, Figure 1) or 12N4 macrocycle (C-177, Figure 1) (Meares et al, 1984). The remaining mixture was desalted into 0.1 M sodium acetate buffer pH 5.0.…”

Section: Materis and Methodsmentioning

confidence: 99%

“…The chelation of "'In by conjugates of this type is relatively weak, and the use of these in vivo has led to significant instability of the labelled conjugate in both animal model studies and patients (Hnatowich et al, 1985). This instability is particularly manifested by the accumulation of high levels of "'In in the liver (Shumacher et al, 1990), possibly as a result of release of free "'In, which is then bound by serum transferrin and transported to the liver (Meares et al, 1984). Recently, novel bifunctional derivatives of DTPA have been devised in which the carbon backbone of the chelate has a linking group inserted for attachment to the antibody such that all eight coordination sites on DTPA are preserved (Brechbiel et al, 1986).…”

mentioning

confidence: 99%

See 1 more Smart Citation

Comparative biodistributions of indium-111-labelled macrocycle chimeric B72.3 antibody conjugates in tumour-bearing mice

Turner

King²,

Farnsworth³

et al. 1994

Br J Cancer

View full text Add to dashboard Cite

S_inmary A novel "'In ligand (a C-functionalised derivative of 1,4,7-triazacyclononanetriacetic acid), termed 9N3, was covalently attached to chimeric B72.3, labelled with "'In and compared with ".In-labelled chimeric B72.3 diethylenetriaminepentaacetic acid (DTPA) cyclic anhydnde conjugate (cDTPA) and a C-linked derivative of DTPA (CT-DTPA) in athymic mice bearing human colon carcinoma xenografts. Significant differences in biodistribution were observed between 9N3 and cDTPA conjugates especially in the tumour uptake and blood, liver, femur and colon levels at 24, 48 and 144 h. Significantly higher tumour uptake was observed for "'In-cB72.3-9N3 compared with "'.In-cB72.3-cDTPA at all time points. Radiolocalisation (RI) indices increased with time for the 9N3 conjugate but remained constant for the cDTPA conjugate. The biodistribution of "'In-labelled cB72.3-CT-DTPA was similar to that of ".In-labelled cB72.3-9N3 except for elevated kidney levels. A 12N4 macrocycle (a C-functionalised derivative of 1,4,7,10-tetraazacyclododecanetetraacetic acid) was also tested for its ability to chelate "'In and its biodistribution examined. Labelled conjugates with this macrocycle were more difficult to prepare in a stable form but gave a very similar biodistribution to the 9N3 macrocycle conjugate. Macrocycle-antibody conjugates of this type offer considerable promise for tumour imaging in patients.

show abstract

Conjugation of antibodies with bifunctional chelating agents: Isothiocyanate and bromoacetamide reagents, methods of analysis, and subsequent addition of metal ions

Cited by 335 publications

References 21 publications

Improved radiotracing of oxytocin receptor-expressing tumours using the new [111In]-DOTA-Lys8-deamino-vasotocin analogue

Improved radiotracing of oxytocin receptor-expressing tumours using the new [111In]-DOTA-Lys8-deamino-vasotocin analogue

Site-specific Conjugation on Serine → Cysteine Variant Monoclonal Antibodies

Comparative biodistributions of indium-111-labelled macrocycle chimeric B72.3 antibody conjugates in tumour-bearing mice

Contact Info

Product

Resources

About