In Vitro Cytotoxicity and In Vivo Anti-Tumor Efficacy of CD13 Targeted Peptide-Monomethyl Auristatin E (MMAE) Conjugates

A series of novel peptide ligands that specifically bind to tumor vascular endothelial CD13 receptor were designed, synthesized and evaluated for in vitro binding. In this study, peptide-monomethyl auristatin E (MMAE) conjugates were evaluated for their in-vitro cytotoxicity and mice in-vivo anti-tumor efficacy. The peptides [PEP20, GYPAY; and PEP173 GYPAVYLF] were synthesized by standard solid phase peptide synthesis method. The drug-linker conjugate maleimidocaproyl-valine-citrulline-paminobenzoyloxycarbonyl-monomethyl auristatin E (mc-vc-PABC-MMAE) was used to prepare the peptide-drug conjugates (PDCs). Formation of the PDCs was confirmed by ESI-MS (PEP20-MMAE, 1062.5 [M+H+Na]⁺²; PEP173-MMAE, 828.9 [M+2H+Na]⁺³) and the % purities of the PDCs after purification were higher than 98%. For invitro cytotoxicity study, CD13 +ve HT1080, CD13-ve MCF7 and HEK293 (normal cells) cells were incubated with various concentrations of PDCs/MMAE. The drug, MMAE, showed very high potency (low IC₅₀ values) across all three cell lines (HT-1080 cells, IC₅₀ 0.09358 nM; MCF-7 cells, IC₅₀ 0.4250 nM; HEK-293 cells, IC₅₀ 0.8354 nM). PDCs (PEP20-MMAE and PEP173-MMAE) showed significantly lower cytotoxicity than MMAE in all cell lines. PEP20-MMAE showed 5.2 and 4.3 times lower cytotoxicity in CD13 negative MCF-7 and control normal HEK-293 cells, respectively, when compared to that in CD13 positive HT-1080 cells. PEP173-MMAE was found to have approximately 2.4 times less cytotoxicity in both MCF-7 cells and HEK-293 cells as compared to HT-1080 cells. For the anti-tumor efficacy study, 975 nmol/kg, which is equivalent to 0.70 mg/kg of MMAE, was selected as the treatment dose. In the mice treated with only PBS, the tumor grew rapidly and reached approximately 450 mm³ by day 28 after tumor implantation (Figure 5). MMAE, PEP20-MMAE, and PEP173-MMAE, all showed almost complete tumor regression during the study. PEP20-MMAE and PEP173-MMAE showed slightly higher tumor regression than MMAE, but the difference was not statistically significant. However, the PDCs exhibited much lower weight loss in mice as compared to the drug MMAE indicating lower side effects in vivo. The limited effectiveness of peptide drug conjugates in in vivo mice tumor model suggested the need of further research to achieve the optimal chemical configuration of the conjugates for in vivo targeting.