A Novel NAMPT Inhibitor-Based Antibody-Drug Conjugate Payload Class for Cancer Therapy

Abstract

Inhibition of intracellular nicotinamide phosphoribosyltransferase (NAMPT) represents a new mode of action for cancer-targeting antibody-drug conjugates (ADCs) with activity also in slowly proliferating cells. To extend the repertoire of available effector chemistries, we have developed a novel structural class of NAMPT inhibitors as ADC payloads. A structure-activity relationship-driven approach supported by protein structural information was pursued to identify a suitable attachment point for the linker to connect the NAMPT inhibitor with the antibody. Optimization of scaffolds and linker structures led to highly potent effector chemistries which were conjugated to antibodies targeting C4.4a (LYPD3), HER2 (c-erbB2), or B7H3 (CD276) and tested on antigen-positive and -negative cancer cell lines. Pharmacokinetic studies, including metabolite profiling, were performed to optimize the stability and selectivity of the ADCs and to evaluate potential bystander effects. Optimized NAMPTi-ADCs demonstrated potent in vivo antitumor efficacy in target antigen-expressing xenograft mouse models. This led to the development of highly potent NAMPT inhibitor ADCs with a very good selectivity profile compared with the corresponding isotype control ADCs. Moreover, we demonstrate─to our knowledge for the first time─the generation of NAMPTi payload metabolites from the NAMPTi-ADCs in vitro and in vivo. In conclusion, NAMPTi-ADCs represent an attractive new payload class designed for use in ADCs for the treatment of solid and hematological cancers.

Figure 1

X-ray cocrystal structure of compound (−)-1 in complex with the hNAMPT protein. The structure was determined at 1.86 Å resolution (PDB accession code 7PPE).

Figure 2

Metabolism of NAMPTi-ADCs with EC3. (A) In vitro formation of metabolites in C4.4a-expressing and mock A549 cells after treatment with C4.4a-EC3 or isotype control-EC3 for 24 h, expressed as relative mass spectrometric area response after HPLC-MS analysis. Medium was used as the control. Stars indicate that the specific metabolites were below the limit of detection. (B) In vivo formation of the phosphoribosylated metabolite P-20 in tumor and liver of THP-1 tumor-bearing mice after treatment with B7H3-EC3 or isotype control-EC3 [5 mg/kg, Q3/4Dx3, intravenous (i.v.)] for 24 h. (C) Structures of EC3-ADC metabolites. [O] in compound P-1 indicates oxidation of the bicyclic ring.

Figure 3

Optimized ECs (A) EC4 and (B) EC5. EC4 was used for the NAMPTi-ADCs that were tested in vivo.

Figure 4

In vivo antitumor efficacy of C4.4a-, HER2-, and B7H3-targeted NAMPTi-ADCs. (A) Growth curves of THP-1 tumors in female scid mice (n = 7/group) treated with vehicle, B7H3-EC3 (DAR 4.3 or 7.8), or isotype control-EC3 (5 mg/kg, i.v., Q7Dx3), as indicated by black arrows. (B) Growth curves of MDA-MB-453 tumors in female NOD-scid mice (n = 7–10 mice/group) treated with vehicle, C4.4a-EC4, HER2-EC4, or isotype control-EC4 (10 mg/kg, i.v., Q7Dx7), as indicated by black arrows. Statistical analyses were performed using the estimated linear model corrected with Sidak’s or Tukey’s method. i.v., intravenously, Q7D, every 7 days; ***, P < 0.001 vs vehicle; ^##, P < 0.01; and ^###, P < 0.001 vs isotype control.