PF231 REDIRECTED CYTOTOXICITY AND PROLIFERATION OF T CELLS INDUCED BY BITE® ANTIBODY CONSTRUCTS ARE MODULATED BY THE EXPRESSION PROFILE OF COSTIMULATORY AND COINHIBITORY MOLECULES ON TARGET CELLS IN VITRO
Authors: Marcinek A. , Brauchle B. , Kischel R. , Kufer P. , Bücklein V. , Metzeler K. , von Bergwelt M. , Spiekermann K. , Subklewe M. 
Published: June 2019

Background:

BiTE® antibody constructs represent a novel immunotherapeutic strategy relying on the recruitment of T cells against tumor cells independent of TCR specificity. In Acute Myeloid Leukemia (AML), CD33 represents a validated target antigen with high prevalence in expression in >90 % of primary AML samples (Krupka et al. 2014). A CD33 specific BiTE® antibody construct was developed (AMG 330) and showed cytotoxicity against primary AML in vitro (Krupka et al. 2016). AMG 330 is being evaluated in a ongoing dose escalation of a first in human phase 1 study in relapsed/refractory AML with encouraging early evidence of tolerability and anti‐leukemic activity in heavily pretreated patients (Ravandi et al. 2018). To better understand molecular mechanisms mediating AMG 330 effects, we created a novel in vitro model system to characterize the effects of costimulatory and coinhibitory molecules on redirected cytotoxicity and induced proliferation of T cells.

Aims:

We hypothesized that BiTE®‐mediated cytotoxicity and induction of T‐cell proliferation is influenced by the checkpoint expression profile on target cells. We assume that the ratio of positive and negative checkpoint molecules on target cells is an important modulator of AMG 330‐mediated cytotoxicity against AML cells.

Methods:

To dissect mechanistic aspects of T cell recruitment a stable expression system was established utilizing murine Ba/F3 cells expressing human CD33  ±  CD80  ±  CD86  ±  PD‐L1. Co‐cultures of murine Ba/F3 transfectants and human healthy donor (huHD) T cells were performed in presence of AMG 330 or a control BiTE® (cBiTE®) (5 ng/ml). For some experiments, huHD T cells were separated into naive (CD45RA+/CCR7+) vs memory (CD45RADIM) cells using fluorescence‐activated cell sorting. Further co‐cultures were performed by mixing varying proportions of Ba/F3 CD33+ CD80+ PD‐L1 ±  and Ba/F3 CD33+ PD‐L1+ cells with huHD T cells (AMG 330/cBite c = 0.5 ng/ml). Proportion of Ba/F3 CD33+ PD‐L1+ cells was varied between 0–100 %. After 3 days, specific lysis was determined by flow cytometry and calculated as % specific lysis = 100 × (1–live CD33+ cellsAMG
330/live CD33+ cellscBiTE). T‐cell proliferation was defined as number of CD2+ cells on day 3 compared to day 0.

Results:

Murine Ba/F3 cells expressing only human CD33 were not lysed upon addition of AMG 330 and huHD T cells. Cytotoxicity could be observed with cells additionally expressing human CD80+CD86 >> CD80 > CD86 (Table 1, A). There was a direct correlation of AMG 330 mediated T‐cell proliferation and cytotoxicity. Memory T cells showed increased cytotoxicity compared to naive T cells against Ba/F3 cell lines expressing the different sets of human molecules. The influence of co‐inhibition was investigated by additionally transducing PD‐L1 into the different Ba/F3 cells. This led to a reduced AMG 330‐mediated cytotoxicity in all PD‐L1 expressing Ba/F3 cells and was accompanied by a reduction in T‐cell proliferation (Table 1, A). When mixing Ba/F3 CD33+ CD80+ PD‐L1 ±  with Ba/F3 CD33+ PD‐L1+ cells we observed a decrease in T‐cell proliferation and mediated cytotoxicity with increased percentage of Ba/F3 CD33+ PD‐L1+ cells (Table 1, B).

Summary/Conclusion:

In summary, this data supports the hypothesis T‐cell proliferation and thereto related cytotoxicity induced by redirection through BiTE® antibody constructs are influenced by the expression of costimulatory and coinhibitory molecules. Our data support the notion that the checkpoint profile on AML and its distribution among target cells, rather than one molecule by itself on a single cell level, may modulate T‐cell response to AMG 330.

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