Our investigations continued with determination of the stabi

Our investigations continued with determination of the stability and reactivity of the two componen
Our investigations continued with determination of the
stability and reactivity of the two components in biological
media.In vitro assays in PBS,serum,and blood showed
(Table 1) that [111In]-1 should be stable for the duration of its
presence in mice (blood clearance half-life of 9.8 minutes; see
Figure S13 in the Supporting Information).We observed a
fast reaction between [111In]-1 and CC49-TCO in vitro in
semi-equimolar conditions and at low concentration (3.3 mm)
within 10 minutes in PBS,serum and blood (Table 2).The
yields obtained from the reactions with 10 and 15 equivalents
of [111In]-1 indicated the presence of an average of 7.4 reactive
TCO moieties per antibody.Furthermore,the nonspecific
binding of [111In]-1 to unmodified CC49 or other media
constituents was found to be very low.To assess the stability
of the mAb-bound TCO moiety in vivo we treated mice with
CC49-TCO and then treated extracted blood samples ex vivo
with an excess of tetrazine 1.The reaction yield,corrected for
CC49-TCO blood clearance,revealed that 75% of the CC49-
bound TCO present in blood was still reactive after 24 hours
circulation,thus indicating good in vivo stability.
To test the Diels–Alder reaction in living animals we
administered CC49-TCO to mice bearing colon cancer
xenografts,followed one day later with injection of 3.4 equivalents
of [111In]-1 with respect to TCO.The chemically tagged
tumors reacted rapidly with [111In]-1,resulting in pronounced
localization of radioactivity in the tumor,as demonstrated by
single photon emission computed tomography/computed
tomography (SPECT/CT) imaging of live mice three hours
after injection (Figure 3a,d; see movie S1 in the Supporting
Information).The SPECT quantification (see Table S3 in the
Supporting Information) of the tumor gave 4.2% injected
dose per gram (%IDg\31) and a tumor-to-muscle ratio (T/M)
of 13.1:1.We also observed limited uptake in blood and
nontarget tissues,such as the liver,which we attributed to

我们的研究随着确定在生物媒质中两种成分的稳定性和反应性而继续.在PBS、血清和血中的体外化验表明（表1）[IIIIn]-1对于其存在于老鼠中的持续时间（血液清除率半衰期为9.8min；见支持信息中的图S13）来说是稳定的.我们观察到在[IIIIn]-1和CC49-TCO之间在半等摩尔条件下和低浓度（3.3μM）下,在PBS、血清和血中,于10min内快速的体外反应（表2）.与10和15个当量的[IIIIn]-1反应所得到的产率表明,每个抗体存在平均7.4个反应性TCO分子部分.而且,我们发现[IIIIn]-1与未改性CC49或其他媒质组分的非特异性结合是非常低的.为了估计mAb束缚的TCO分子部分在体内的稳定性,我们用CC49-TCO用处理老鼠,然后用过多的四嗪1体外处理抽取的血液样本.针对CC49-TCO血液清除率修正的反应产率揭示,75%的存在于血液中的CC49束缚的TCO在24h循环后仍是反应性的,因此表明了体内稳定性的良好.
为了测试在活的动物中的Diels-Alder反应,我们将CC49-TCO给药给承受结肠癌异种移植物的老鼠,一天后在注射相对于TCO的3.4当量的[IIIIn]-1.该加化学标记的肿瘤与[IIIIn]-1迅速反应,导致放射性在肿瘤中明显的局部化（定位）,就像活老鼠在注射后3h由单一质子发射计算的层析X射线照相法/计算的层析X射线照相法（SPECT/CT）成像所演示的那样（图3a,d；见支持信息中的电影S1）.此肿瘤的SPECT量化（见支持信息中的表S3）给出了每克4.2%的注射剂量（%IDg－1）和13.1：1的肿瘤与肌肉比率（T/M）.我们还观察到在血液和非目标组织,例如肝脏中有限的摄取,对这,我们归因于