Autologous T Cells From AML Patients Can Be Effectively Recruited for In-Vitro Lysis of Blasts by a Novel CD33/CD3-Bispecific BiTE Antibody

Abstract 14

Bispecific T cell-engaging (BiTE®) antibodies combine in one polypeptide chain two single chain antibodies, one specific for CD3 on T cells and one for a tumor-associated antigen. The CD19/CD3-bispecific BiTE antibody blinatumomab has shown in phase 1 and 2 clinical trials very high response rates in patients with non-Hodgkin's lymphoma and acute lymphoblastic leukemia. Here, we report on the potential of a novel BiTE antibody targeting CD33, an antigen broadly expressed by myeloid cells including acute myelogenous leukemia (AML) blasts, in redirecting autologous T cells for in vitro lysis of blasts from AML patients.

In a first step, the cytolytic potential of the CD33-specific BiTE (CD33 BiTE) was investigated in co-cultures of enriched resting CD8⁺ T cells from healthy donors and CD33⁺ leukemic cell lines KG-1 and U-937 as target cells. CD33 BiTE concentrations as low as 0.1 ng/ml (1.8 pM) mediated effective lysis of leukemic cell lines at effector to target (E:T) ratios of 1:1, whereas no lysis was observed with a solely CD3-binding control BiTE antibody. Peripheral CD8⁺ T cells that were pre-activated in cell culture or CD8⁺ T cell clones were even more potent in target cell lysis than previously resting T cells. Data obtained with a ⁵¹Cr release assay were comparable to those from a flow cytometry-based assay. Next, primary samples from AML patients were co-cultured with mononuclear cells (MNC) from healthy donors at an E:T ratio of 1:1. After 48 hrs of incubation in the presence of 1 ng/ml CD33 BiTE, a decrease in CD33⁺ AML blasts as well as of CD33⁺ monocytes was observed when compared to samples with control BiTE or vehicle. The CD33 BiTE induced upregulation of activation markers CD25 and CD69 on the majority of T cells. We furthermore investigated whether T cells from AML patients were capable of mediating lysis of CD33⁺ leukemia cells by CD33 BiTE. Resting or in vitro pre-stimulated CD8⁺ T cells were prepared from peripheral blood of newly diagnosed AML patients and tested for lysis of U937 target cells. Redirected T cells from AML patients were capable of eliminating leukemic cells in the presence of CD33 BiTE as effectively as T cells from healthy controls.

Finally, we developed a FACS-based assay that allowed studying autologous blast lysis and T cell behaviour using cryo-preserved patient samples. Upregulation of T cell activation markers in cultures of MNC samples from AML patients was evident following addition of 1 ng/ml CD33 BiTE. Fifty five and 85% of CD4⁺ cells, and 57 and 65% of CD8⁺ cells expressed CD25 after 24 h and 48 h, respectively, but not with the control BiTE antibody (all <6%). Despite robust T cell activation, only a limited lysis of myeloid blasts was observed, presumably, due to the short incubation periods and low E:T ratios in the range of 1:5-1:21. We therefore investigated whether blast lysis is more effective after prolonged incubation. In the presence of CD33 BiTEs for 6 days, T cell numbers in AML patient samples dramatically expanded; CD8⁺ cell counts were up 8-fold, and CD4⁺ cell counts up 11-fold. This was not observed under control conditions. Up to 85% of AML blasts were now lysed. Currently, a larger collection of primary AML patient samples is being analyzed in order to determine an ex-vivo response rate for CD33 BiTE treatment and the impact of the patient samples’ E:T ratio and CD33 expression level on blasts on redirected lysis.

Taken together, the novel CD33 BiTE effectively engages and activates autologous T cells for the elimination of AML blasts in vitro and may thereby constitute a novel therapeutic option for the treatment of patients with CD33-expressing myeloid leukemia.