Reaction SMILES: [CH3:1][CH2:2][C@@H:3]([C@H:5]([N:35]([C:37]([C@@H:39]([NH:43][C:44]([C@@H:46]([N:50]([C:52]([O:54][CH2:55][C:56]1[CH:57]=[CH:58][C:59]([NH:62][C:63]([C@@H:65]([NH:73][C:74]([C@@H:76]([NH:80][C:81]([CH2:83][CH2:84][CH2:85][CH2:86][CH2:87][N:88]2[C:93](=[O:94])[CH:92][CH2:91][C:89]2=[O:90])=[O:82])[CH:77]([CH3:79])[CH3:78])=[O:75])[CH2:66][CH2:67][CH2:68][NH:69][C:70]([NH2:72])=[O:71])=[O:64])=[CH:60][CH:61]=1)=[O:53])[CH3:51])[CH:47]([CH3:49])[CH3:48])=[O:45])[CH:40]([CH3:42])[CH3:41])=[O:38])[CH3:36])[C@H:6]([O:33][CH3:34])[CH2:7][C:8]([N:10]1[C@H:14]([C@H:15]([O:31][CH3:32])[C@H:16]([C:18]([NH:20][C@@H:21]([C@@H:23]([OH:30])[C:24]2[CH:25]=[CH:26][CH:27]=[CH:28][CH:29]=2)[CH3:22])=[O:19])[CH3:17])[CH2:13][CH2:12][CH2:11]1)=[O:9])[CH3:4].[NH2:95][C@H:96]([C:100]([NH:102][C@H:103]([C:111]([OH:113])=[O:112])[CH2:104][CH2:105][CH2:106][NH:107][C:108]([NH2:110])=[O:109])=[O:101])[CH:97]([CH3:99])[CH3:98].CC[C@@H]([C@H](N(C([C@@H](NC([C@@H](N(C(OCC1C=CC(NC([C@@H](NC([C@@H](NC(CCCCCN2C(=O)[CH]CC2=O)=O)C(C)C)=O)CCCNC(N)=O)=O)=CC=1)=O)C)C(C)C)=O)C(C)C)=O)C)[C@H](OC)CC(N1[C@H]([C@H](OC)[C@H](C(N[C@@H]([C@@H](O)C2C=CC=CC=2)C)=O)C)CCC1)=O)C.C(S)[C@@H](O)[C@H](O)CS.[NH2:216][C@H:217]([C:220](O)=[O:221])[CH2:218][SH:219]>CS(C)=O>[NH2:216][C@H:217]([C:220]([NH:95][C@H:96]([C:100]([NH:102][C@H:103]([C:111]([OH:113])=[O:112])[CH2:104][CH2:105][CH2:106][NH:107][C:108]([NH2:110])=[O:109])=[O:101])[CH:97]([CH3:98])[CH3:99])=[O:221])[CH2:218][SH:219].[CH3:1][CH2:2][C@@H:3]([C@H:5]([N:35]([C:37]([C@@H:39]([NH:43][C:44]([C@@H:46]([N:50]([C:52]([O:54][CH2:55][C:56]1[CH:57]=[CH:58][C:59]([NH:62][C:63]([C@@H:65]([NH:73][C:74]([C@@H:76]([NH:80][C:81]([CH2:83][CH2:84][CH2:85][CH2:86][CH2:87][N:88]2[C:93](=[O:94])[CH:92][CH2:91][C:89]2=[O:90])=[O:82])[CH:77]([CH3:78])[CH3:79])=[O:75])[CH2:66][CH2:67][CH2:68][NH:69][C:70]([NH2:72])=[O:71])=[O:64])=[CH:60][CH:61]=1)=[O:53])[CH3:51])[CH:47]([CH3:49])[CH3:48])=[O:45])[CH:40]([CH3:42])[CH3:41])=[O:38])[CH3:36])[C@H:6]([O:33][CH3:34])[CH2:7][C:8]([N:10]1[C@H:14]([C@H:15]([O:31][CH3:32])[C@H:16]([C:18]([NH:20][C@@H:21]([C@@H:23]([OH:30])[C:24]2[CH:25]=[CH:26][CH:27]=[CH:28][CH:29]=2)[CH3:22])=[O:19])[CH3:17])[CH2:13][CH2:12][CH2:11]1)=[O:9])[CH3:4]
Reactant: N[C@@H](CS)C(=O)O
Reactant: CC(C)[C@H](N)C(=O)N[C@@H](CCCNC(N)=O)C(=O)O
Agent: CC[C@H](C)[C@@H]([C@@H](CC(=O)N1CCC[C@H]1[C@H](OC)[C@@H](C)C(=O)N[C@H](C)[C@@H](O)c1ccccc1)OC)N(C)C(=O)[C@@H](NC(=O)[C@H](C(C)C)N(C)C(=O)OCc1ccc(NC(=O)[C@H](CCCNC(N)=O)NC(=O)[C@@H](NC(=O)CCCCCN2C(=O)[CH]CC2=O)C(C)C)cc1)C(C)C
Agent: O[C@H](CS)[C@H](O)CS
Solvent: CS(C)=O
Product: CC(C)[C@H](NC(=O)[C@@H](N)CS)C(=O)N[C@@H](CCCNC(N)=O)C(=O)O
Temperature: 10 °C
Reaction time: 2 h

Procedure:
The generation of cAC10 ADCs with two and four MMAE molecules per antibody involved a partial reduction followed by reaction with Val-Cit-MMAE. The antibody cAC10 (10 mg/ml) was partially reduced by addition of DTT to a final DTT:mAb molar ratio of 3.0 followed by incubation at 37° C. for ˜2 h. The reduction reaction was then chilled to ˜10° C. and the reduced cAC10 purified away from excess DTT via diafiltration. Following diafiltration, the thiol concentration in the partially-reduced cAC10 was determined by the DTNB (Ellman's) assay; in this manner, an average of about 2 disulfide bonds were reduced, thus exposing about 4 reduced Cys:mAb. To conjugate all of the reduced Cys, Val-Cit-cMMAE was added to a final Val-Cit-MMAE:reduced Cys molar ratio of about 1.15. The conjugation reaction was carried out in the presence of 15% v/v of DMSO and allowed to proceed at about 10° C. for about 30 min. Following the conjugation reaction, excess free Cys (2 moles of Cys per mole of Val-Cit-MMAE) was added to quench unreacted Val-Cit-MMAE to produce the Cys-Val-Cit-MMAE adduct. The Cys quenching reaction was allowed to proceed at about 10° C. for about 30 min. The Cys-quenched reaction mixture was purified and buffer-exchanged into PBS by diafiltration to obtain the partially loaded cAC10-Val-Cit-MMAE.