Three rationally designed pyrrolobenzodiazepine (PBD) drug-linkers have been synthesized via intermediate 19 for use in antibody-drug conjugates (ADCs). They lack a cleavable trigger in the linker and consist of a maleimide for cysteine antibody conjugation, a hydrophilic spacer, and either an alkyne (6), triazole (7), or piperazine (8) link to the PBD. In vitro IC50 values were 11-48 ng/mL in HER2 3+ SK-BR-3 and KPL-4 (7 inactive) for the anti-HER2 ADCs (HER2 0 MCF7, all inactive) and 0.10-1.73 μg/mL (7 inactive) in CD22 3+ BJAB and WSU-DLCL2 for anti-CD22 ADCs (CD22 0 Jurkat, all inactive at low doses). In vivo antitumor efficacy for the anti-HER2 ADCs in Founder 5 was observed with tumor stasis at 0.5-1 mg/kg, 1 mg/kg, and 3-6 mg/kg for 6, 8, and 7, respectively. Tumor stasis at 2 mg/kg was observed for anti-CD22 6 in WSU-DLCL2. In summary, noncleavable PBD-ADCs exhibit potent activity, particularly in HER2 models.