Development of a Bifunctional Checkpoint Inhibitory T Cell Engager (CiTE) to Reverse Adaptive Immune Escape in AML
Authors: Herrmann Monika , Krupka Christina , Deiser Katrin , Lindl Bettina , Mocikat Ralph , Metzeler Klaus H. , Spiekermann Karsten , Kobold Sebastian , Fenn Nadja , Hopfner Karl-Peter , Subklewe Marion 
Published: October 2019

Abstract
The CD33-targeting bispecific T cell engager (BiTE®) AMG 330 proved to be highly efficient in mediating cytotoxicity of AML cells in vitro and in mouse models (Krupka et al, Blood 2014). Yet, T cell activation is correlated with the upregulation of PD-L1 and other inhibitory checkpoint molecules on AML cells that confer adaptive immune resistance (Krupka et al, Leukemia 2016). PD-1/PD-L1 blocking agents may counteract T cell dysfunction, however, at the expense of broadly distributed immune-related adverse events (irAEs). We developed a checkpoint inhibitory T cell engaging (CiTE) antibody that combines T cell redirection to CD33 on AML cells with locally restricted immune checkpoint blockade.
CiTE constructs were generated by first fusing a high-affinity CD33 single-chain variable fragment (scFv) to a CD3ε scFv in one polypeptide chain. Next, this single-chain chain was fused to the extracellular domain of PD-1 (PD-1ex), which naturally holds a low affinity to PD-L1. Antigen binding of CiTE constructs as well as CiTE mediated cytotoxicity of AML cell lines and primary AML cells were done using multiparameter flow cytometry. T cell activation and cytotoxicity assays were complemented by cytometric bead arrays. Murine AML xenograft studies using non-obese diabetic (NOD) scid gamma mice were used for engraftment of primary AML cells and assessment of CiTE mediated cytotoxicity in vivo.
CiTE antibody constructs were successfully generated by fusing the bispecific CD33-CD3ε scFv to the endogenous extracellular domain of human PD-1 (PD-1ex). The CiTE was compared to a single chain triplebody (sctb), in which PD-1ex was replaced by a high-affinity PD-L1 scFv. The BiTE-like molecule, PD-1ex.αCD3 and αPD-L1.αCD3, as well as a non-targeting molecule served as controls. When investigating CiTE and sctb as whole molecules, both bound with similar affinities to CD33+PD-L1+ AML cell lines and HD T cells. CiTE- and sctb-induced upregulation of CD69 and CD25 on healthy donor T cells in the presence of MOLM-13-PD-L1 cells. By a synergistic effect of checkpoint blockade and avidity-dependent binding, the PD-1ex attachment increased T cell activation (3.3-fold elevation of IFN-γ release) and lead to efficient and highly selective cytotoxicity of CD33+PD-L1+ cells (EC50 = 2.3 pM to 26.9 pM) as well as primary AML patient samples (n=8). CiTE induced preferential lysis of CD33+PD-L1+ cells and had no activity against CD33-PD-L1+ cells. This was supported by the observation that the CiTE molecule was able to selectively induce elimination of CD33+PD-L1+ cells in the presence of PD-L1+ cells. In a murine xenograft model, the CiTE induced complete AML eradication without causing leukemia-unrelated T cell activation or body weight loss. Notably, murine and human PD-L1 bind with similar affinities to PD-1.
We conclude that our molecule preferentially targets CD33+PD-L1+ AML cells, whereas high-affinity blocking agents also address PD-L1+ non-AML cells. Based on these findings, we expect to reverse adaptive immune escape mechanisms of T cell recruiting antibody formats and avoid irAEs associated with systemic checkpoint blockade, suggesting efficient therapeutic potential particularly for patients with relapsed or refractory AML. Future studies will need to further examine efficiency and tolerance in advanced in vivo models before applying the CiTE format into a clinical setting.

Disclosures
Lindl: Amgen: Research Funding. Metzeler:Celgene: Consultancy, Research Funding; Novartis: Consultancy. Subklewe:Gilead: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria; Roche: Consultancy, Research Funding; Celgene: Consultancy, Honoraria.