Figure 1.
Structures of (DOTA)-decalysine, (DOTA)3-decalysine, and (DOTA)5-decalysine.
Figure 2.
LC-ESI-TOF mass spectrometry analysis of the heavy chain (HC) of the used antibodies.
Deconvolution of the raw data was accomplished by MaxEnt1. A. Mass spectrum of HC for chCE7degl (left) and chCE7agl (right). B. Mass spectrum of HC for chCE7degl coupled to (DOTA)-decalysine (left) and chCE7agl coupled to (DOTA)-decalysine (right); C. Mass spectrum of HC for chCE7degl coupled to (DOTA)3-decalysine (left) and chCE7agl coupled to (DOTA)3-decalysine (right).
Figure 3.
Enzymatic conjugation of (DOTA)n-decalysine to chCE7degl or chCE7agl, an aglycosylated variant of the antibody.
Although there are two enzymatic recognition sites per heavy chain of chCE7agl (Gln295 and Gln297), only one molecule of (DOTA)n-decalysine (n = 1, 3, 5) was conjugated using transglutaminase. Interestingly, two isopeptide bonds were formed between one substrate and one heavy chain, as indicated by the loss of two ammonia molecules (bottom).
Table 1.
Mass spectrometry results of the unmodified and modified heavy chains (HC).
Table 2.
General in vitro and in vivo features of chCE7agl-[(DOTA)n-decalysine]2 (n = 1, 3, 5).
Table 3.
Biodistribution of 177Lu-labeled chCE7agl-[(DOTA)n-decalysine]2 in nude mice with human ovarian cancer (SKOV3ip) metastases.
Figure 4.
Isoelectric focusing of chCE7agl and chCE7agl-[(DOTA)n-decalysine]2 on an IEF agarose plate pH 3–10.
Samples with a concentration of 5 µg/µL were dialyzed against deionized water before loading and 5 µL of each probe were loaded 4 cm from the cathode. After prefocusing at 1 W constant power for 10 min and focusing for 90 min (20 W power limit; 1000 voltage limit) the gels were fixed in 30% TCA and proteins were stained with Coomassie. A. Lane 1: IEF standard Biorad No 161-0310; lane 2: The parent antibody chCE7agl revealed a pI of 7.8. B. Lane 1: IEF marker Serva No 39212.01. The (DOTA)n-decalysine conjugates showing anodal movement depending on the numbers of DOTA chelats coupled to the decalysine. Lane 2: chCE7agl-[(DOTA)-decalysine]2; lane 3: chCE7agl-[(DOTA)3-decalysine]2; lane 4: chCE7agl-[(DOTA)5-decalysine]2.