Recent immunotherapeutic approaches designed to augment T- and B-cell mediated killing of tumor cells has met with clinical success suggesting that immuno-oncology (IO) approaches have tremendous potential for treatment in a broad spectrum of tumor types. After complex recognition of target cells by T and B cells, delivery of the serine protease granzyme B (GrB) to tumor cells comprises the cytotoxic insult resulting in a well-characterized, multimodal apoptotic cascade. We designed a recombinant fusion construct composed of a human anti-Her2 scFv fused to active GrB for recognition and delivery of GrB to tumor cells simulating T and B-cell therapy. The GrB-Fc-4D5 dimeric construct (mw 160 kDa) was generated and expressed in stably-transfected CHO-S cells at ~60 mg/L and purified to homogeneity. The enzymatic activity of the fusion construct was similar to commercially-available GrB and the affinity of the construct for purified Her2 extracellular domain (ECD) was 0.328 nM, comparable to that of Herceptin (0.150 nM). The GrB-Fc-4D5 construct was highly cytotoxic to Her2-positive cells such as SKBR3, MCF7 and MDA-MB-231 with IC50 values of 56, 99 and 27 nM respectively. Using immunofluorescence, the fusion construct internalized rapidly into target (SKBR3 or SKOV3) cells within 1 h of exposure, rapidly delivering GrB to the cytoplasmic compartment in a similar manner to that of immune T and B-cell targeting but without the action of the transmembrane pore-forming agent perforin. Against a large panel of various tumor types, GrB-Fc-4D5 was highly cytotoxic to virtually all cells regardless of natural expression levels of the nominal endogenous GrB inhibitor PI-9. Contemporaneous studies with Kadcyla demonstrate similar levels of in vitro activity against virtually all HER2-positive cells tested. GrB-Fc-4D5 demonstrated activity against both log-phase and confluent tumor cells. In keeping with its relatively high molecular weight (~160 kDa), the construct demonstrated a terminal-phase half-life of 13.6 hrs. In vivo efficacy studies are currently ongoing in several orthotopic xenograft models. Also ongoing are pre-IND toxicology, histopathology and clinical chemistry studies. Research conducted, in part, by the Clayton Foundation for Research.
Citation Format: Lawrence H. Cheung, Yunli Zhao, Khalid A. Mohamedali, Ana Alvarez-Cienfuegos, Walter N. Hittelman, Michael G. Rosenblum. Highly cytotoxic, completely human constructs targeting HER2 and containing the immuno-oncology payload granzyme B [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1646. doi:10.1158/1538-7445.AM2017-1646