Redirection of Immune Effector Cells by Bispecific Antibody Systems for the Treatment of Acute Myeloid Leukemia
Authors: Arndt Claudia , Feldmann Anja , Koristka Stefanie , Michalk Irene , Cartellieri Marc , Stamova Slava , von Bonin Malte , Bornhäuser Martin , Ehninger Gerhard , Bachmann Michael 
Published: October 2019

Abstract
Abstract 1528
Acute myeloid leukemia (AML) is the most common diagnosed cancer affecting the myeloid line of immune cells in adults. Although chemotherapy initially leads to a remission of the disease in 60–80% of the cases, an enormous number of these patients (50–80%) relapse. Hence, new adjuvant therapeutic strategies for the elimination of minimal residual disease (MRD) are needed.
One novel attractive approach is based on recombinant bispecific antibodies (bsAb). BsAb are able to mediate a direct cross-link between T cells and tumor cells. This in turn leads to a polyclonal, MHC- and TCR-independent activation of CD4+ and CD8+ T cells for the effective killing of the recognized target cells. A front runner in this field is the bsAb blinatumomab with dual specificity for CD3 and CD19 which is already used successfully in first clinical trials for the treatment of B-cell lymphomas.
The development of a novel bsAb is usually very time-consuming and includes a series of individual optimization steps to achieve optimal reactivity with lowest possible side effects. The aim of the present work was therefore to establish a novel, more flexible, less time-consuming, modular cell targeting system and to compare its efficiency and sensitivity with classical bsAb.
For the immunotargeting of AML by recombinant antibodies we selected the transmembrane glycoprotein CD33, because 90% of the AML patients have CD33-positive blasts irrespective of the underlying AML-subtype. We developed (i) a novel fully humanized bsAb with dual specificity for CD3 and CD33 and in addition (ii) a novel modular cell targeting system which consists of two different recombinant antibody components (figure 1). The first component (targeting module) is an exchangeable linker module composed of an anti-CD33 single-chain fragment variable (scFv) and a peptide epitope (E). The second component (universal effector module) is a bsAb with antigen binding specificity for the CD3-complex on T cells and for the peptide epitope of the scFv-based linker module. Together both molecules are able to form protein complexes similar to bsAb.
Figure 1: Schematic representation of (i) the direct cross-linking system and (ii) the modular cell targeting system Figure 1:. Schematic representation of (i) the direct cross-linking system and (ii) the modular cell targeting system
The functionality of both immunotargeting systems was examined by performing chromium (51Cr) release assays. As target cells both AML blasts from patients and artificially overexpressing CD33-positive tumor cells were used. Both the direct targeting system consisting of a classical novel humanized anti-CD33-anti-CD3 bsAb and the modular cell targeting system efficiently killed CD33-positive targets cells down to picomolar concentrations (figure 2).
Figure 2: Tumor cell lysis mediated by classical bsAb and the modular cell targeting system was determined by performing a chromium release assay. For this purpose, CD33-positive, 51Cr loaded target cells were incubated with PBMCs as effector cells. Co-cultivation occurred either in the presence or absence of recombinant Ab by using an effector to target cell ratio of 10: 1 and different amounts of recombinant Abs. Figure 2:. Tumor cell lysis mediated by classical bsAb and the modular cell targeting system was determined by performing a chromium release assay. For this purpose, CD33-positive, 51Cr loaded target cells were incubated with PBMCs as effector cells. Co-cultivation occurred either in the presence or absence of recombinant Ab by using an effector to target cell ratio of 10: 1 and different amounts of recombinant Abs.
Compared to the classical bsAb the novel modular cell targeting has a series of advantages: Just by replacing the targeting module the system can be easily modified for retargeting of T cells against other tumor antigens. Moreover, using linker modules with dual specificities enables a multi-specific targeting which may improve the targeting specificity and thereby might increase the success of an immunotherapy.
Taken together, here we present for the first time two novel fully functional humanized, recombinant Ab-based systems for the immunotargeting of CD33+ AML blasts by CD3+ T cells. The obtained data indicate that both the direct cross-linking anti-CD3-anti-CD33 bsAb and the modular cell targeting system hold great potential as an adjuvant cancer immunotherapy for the elimination of MRD in AML-patients.

Disclosures:
No relevant conflicts of interest to declare.