SIB-DOTA: a trifunctional prosthetic group potentially amenable for multi-modal labeling that enhances tumor uptake of internalizing monoclonal antibodies

Abstract

A major drawback of internalizing monoclonal antibodies (mAbs) radioiodinated with direct electrophilic approaches is that tumor retention of radioactivity is compromised by the rapid washout of iodo-tyrosine, the primary labeled catabolite for mAbs labeled via this strategy. In our continuing efforts to develop more versatile residualizing labels that could overcome this problem, we have designed SIB-DOTA, a prosthetic labeling template that combines the features of the prototypical, dehalogenation-resistant N-succinimidyl 3-iodobenzoate (SIB) with DOTA, a useful macrocyclic chelator for labeling with radiometals. Herein we describe the synthesis of the unlabeled standard of this prosthetic moiety, its protected tin precursor, and radioiodinated SIB-DOTA. An anti-EGFRvIII-reactive mAb, L8A4 was radiolabeled with [(131)I]SIB-DOTA in 27.1±6.2% (n=2) conjugation yields and its targeting properties to the same mAb labeled with [(125)I]SGMIB both in vitro and in vivo using U87MG·ΔEGFR cells and xenografts were compared. In vitro paired-label internalization assays showed that the intracellular radioactivity from [(131)I]SIB-DOTA-L8A4 was 21.4±0.5% and 26.2±1.1% of initially bound radioactivity at 16 and 24h, respectively. In comparison, these values for [(125)I]SGMIB-L8A4 were 16.7±0.5% and 14.9±1.1%. Similarly, the SIB-DOTA prosthetic group provided better tumor targeting in vivo than SGMIB over 8 d period. These results suggest that SIB-DOTA warrants further evaluation as a residualizing agent for labeling internalizing mAbs including those targeted to EGFRvIII.

Figure 1

Reversed-phase HPLC profile of isolated [¹³¹I]9

Figure 2

Radioactivity profile from the paired-label size-exclusion HPLC of [¹³¹I]SIB-DOTA-L8A4 and [¹²⁵I]SGMIB-L8A4. Note that IgG elutes with a retention time of about 16-17 min under these conditions.

Figure 3

Results from the paired-label internalization of L8A4 labeled with [¹³¹I]SIB-DOTA (red bars) and that with [¹²⁵I]SGMIB (blue bars) by U87MG·ΔEGFR cells. Cells were incubated with the labeled mAbs at 4°C for 1 h, unbound radioactivity was washed, and the cells were brought up to 37°C. Thereafter, cells were processed as described in the experimental section at various time points to determine the percent of initially bound radioactivity that was internalized (A) and that was in cell culture supernatants (B). Data in C shows the percent of radioactivity in the supernatants that could be precipitated with MeOH.

Figure 4

Tumor uptake of L8A4 labeled with [¹³¹I]SIB-DOTA (red bars) and that with [¹²⁵I]SGMIB (blue bars) after intravenous administration in mice bearing U87MG·ΔEGFR xenografts (n = 5 per time point).

Scheme 1

Synthesis of bis-N-hydroxy succinimidyl 5-iodo-isophthalate

Scheme 2

Synthesis of Bis-N-hydroxy-succinimidyl 5-(tri-n-butylstannyl)isophthalate

Scheme 3

Synthesis of SIB-DOTA standard and the protected tin precursor

Scheme 4

Synthesis radioiodinated SIB-DOTA