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Bispecific T cell engaging (BiTE®) antibody constructs represent a novel class of
therapeutic antibodies, which are comprised of two single-chain variable fragments
simultaneously binding CD3-positive T cells and a specific tumor target antigen 1]. Blinatumomab is the first of BiTE® antibody construct which entered the clinic and
simultaneously binds CD3-positive T cells and CD19-positive B cells. The design of
this BiTE® molecule brings CD19-positive target cells in close contact with CD3-positive
T cells. Importantly, binding of the BiTE® molecule results in polyclonal T cell activation
and expansion which results in effective lysis of the target cells irrespective of
T cell specificity 2], 3]. Blinatumomab has shown to have antileukemic activity in patients with B-precursor
acute lymphoblastic leukemia (ALL) 4], and recently, blinatumomab was approved for the treatment of relapsed or refractory
B-precursor ALL by the FDA (http://www.fda.gov/drugs/informationondrugs/approveddrugs/ucm425597.htm). Loss of CD19 and extramedullary relapse have been observed as mechanisms of resistance
to blinatumomab treatment 5], 6]; however, other mechanisms of resistance have not been reported so far.

Upregulation of programmed death-ligand 1 (PD-L1) on tumor cells in response to endogenous
anti-tumor immunity 7] inhibits adaptive immune responses by inducing T cell dysfunction 8]. Expression of PD-L1 on tumor cells has been associated with poor outcome in solid
cancers 9] as well as hematologic malignancies 10]. Antibodies targeting PD-L1 as well as its receptor on T cells, programmed cell death-1
(PD-1), are being evaluated in a variety of cancers 9], 11], 12], including lymphoid malignancies 7]. Recently, two antibodies targeting PD-1 were granted approval for the treatment
of advanced melanoma (pembrolizumab, nivolumab) as well as metastatic squamous non-small
cell lung cancer (nivolumab). Interestingly, activity of these therapies is not limited
to PD-L1-positive cancers, as clinical responses could be detected in cases with low
PD-L1 expression 13]. There is extensive research in the field trying to develop improved predictive biomarkers
to identify those patients which will response to mono- as well as combination immunotherapies
14].

Case presentation

A 32-year-old male patient presented with refractory B-precursor ALL after frontline
treatment with induction I and II as well as consolidation I of the German multicenter
study group on adult acute lymphoblastic leukemia (GMALL) treatment protocol (analogous
to the GMALL trial 07/2003, Clinicaltrials.gov identifier: 00198991). He was subsequently transferred to our center and received blinatumomab as a continuous
infusion at a dose of 9 ?g/day for 7 days and 28 ?g/day for the subsequent 21 days.
Bone marrow blast count prior to treatment with blinatumomab (baseline) was 30 % (Fig. 1c, left panel), and lymphoblasts homogenously expressed CD19 (Fig. 1a, left panel). Treatment was well tolerated with pyrexia on days 1 through 4 of blinatumomab
treatment being the only adverse event. Examination of peripheral blood revealed a
decrease of total lymphocyte counts during treatment (1596/?l on day 0 vs. 217/?l
on day 28, Fig. 1b) including decreased T cells (960 CD3-positive T cells/?l on day 5 vs. 180/?l on
day 28). Interestingly, there was a moderate increase in CD3-positive T cells within
the bone marrow (5–10 % at baseline vs. 20–30 % after blinatumomab treatment, Fig. 1d).

Fig. 1. Increase of PD-1 and PD-L1 positivity after treatment with blinatumomab. a CD19 vs. CD34 expression of lymphoblasts detected by flow cytometry. Lymphoblasts
showed homogenous expression of CD19 at baseline (pre-treatment) as well as after
blinatumomab treatment (post-treatment). b Lymphocyte counts on peripheral blood during blinatumomab treatment. Lymphocyte counts
decreased during blinatumomab treatment (1596/?l on day 0, 986/?l on day 7, 464/?l
on day 14, 368/?l on day 21, and 217/?l on day 28). c Hematoxylin and eosin stain of paraffin embedded bone marrow core biopsy showing
diffuse infiltration of immature progenitors at both time points (pre-treatment blast
count 30 %, post-treatment 60 %). d Immunohistochemistry of paraffin embedded bone marrow core biopsy stained for CD3
showing spotted infiltration of CD3-positive T cells at baseline (5–10 %, pre-treatment)
and diffuse infiltration after blinatumomab treatment (20–30 %, post-treatment). e Immunohistochemistry of paraffin embedded bone marrow core biopsy stained for PD-1
showing 5 % PD-1-positive cells at baseline (pre-treatment) vs. 15 % after blinatumomab
treatment (post-treatment). f Immunohistochemistry of paraffin embedded bone marrow core biopsy stained for PD-L1
showing 2 % PD-L1-positive blasts at baseline (pre-treatment) vs. 40 % after blinatumomab
treatment (post-treatment)

Upon completion of the first cycle, bone marrow examination revealed persistent leukemia
with a blast count of 60 % (Fig. 1c, right panel), showing homogenous expression of CD19 (Fig. 1a, right panel). Since a loss of CD19 as the mechanism of resistance could not be detected,
we examined PD-1 and PD-L1 expression in the bone marrow by immunohistochemistry.
A moderate increase in PD-1 positivity was seen in lymphocytes in the bone marrow
by immunohistochemistry (5 % PD-1-positive cells at baseline vs. 15 % after blinatumomab
treatment, Fig. 1e). However, we observed a marked increase of PD-L1 positivity (2 % PD-L1-positive
blasts at baseline vs. 40 % after blinatumomab treatment, Fig. 1f). In contrast to the bone marrow, no PD-1 expression was observed on T cells from
the peripheral blood (Fig. 2a).

Fig. 2. Decreased in vitro blinatumomab-mediated lysis of ALL blasts by patient CD3-positive T cells. a PD-1 expression on peripheral CD3-positive T cells was compared to PD-1 expression
on healthy donor CD3-positive T cells showing no detectable difference. b The patient’s ALL blasts were cocultured either with healthy donor CD3-positive T
cells (upper panels) or the patient’s own CD3-positive T cells (lower panels) with either control-Bite® (left panels) or blinatumomab (right panels) and analyzed by flow cytometry after 3 days. c Specific lysis was calculated as one minus the ratio of CD19-positive cells treated
with blinatumomab and CD19-positive cells treated with control-Bite®. Healthy donor
CD3-positive T cells showed efficient lysis of our patient’s ALL blasts (specific
lysis 93.6 %) whereas CD3-positive T cells from our patient showed inefficient lysis
of autologous ALL blasts (specific lysis 8.5 %). d IFN-? concentration in cell culture supernatants were considerably lower for our
patient’s T cells cocultured with his ALL blasts, whereas coculture of healthy donor
T cells and ALL blasts from our patient led to considerable IFN-? production. Cell
cultures with control-Bite® did not show any IFN-? production

Finally, we collected the patient’s peripheral blood after blinatumomab treatment
and purified CD3-positive T cells (EasySepâ„¢ Human CD3 Positive Selection Kit II, Stemcell
Technologies, Vancouver, British Columbia, Canada). Of either patient or healthy donor
CD3-positive T cells, 2.5?×?10
⁵
were cocultured with 5?×?10
⁵
of the patient’s ALL cells (CD3-negative cells) with blinatumomab or control-BiTE®
at a dose of 5 ng/ml on 3?×?10
⁴
irradiated MS-5 feeder cells. After 3 days, cells were stained with CD19-PE, CD2-BV421,
and murine CD29-APC-Cy7 (all BioLegend, San Diego, CA, USA), as well as LIVE/DEAD®
Fixable Aqua Dead Cell Stain Kit (Life Technologies, Carlsbad, CA, USA), and analyzed
on a LSR II flow cytometer (BD Biosciences, Heidelberg, Germany). After cell counting,
the percentage of the respective cell population determined by flow cytometry was
used to determine the absolute cell counts. Percentage of specific lysis was calculated
using cell counts of control-BiTE® antibody-treated relative to blinatumomab-treated
cultures, as described previously 15]. The interferon-? (IFN-?) concentration in cell culture supernatants was measured
by cytometric bead array (BDâ„¢ CBA Human IFN-? Flex Set, BD Biosciences, Heidelberg,
Germany) according to the manufacturer’s instructions on the same machine.

In vitro, the patient’s T cells showed considerably less lysis of CD19-positive cells (8.5
vs. 93.6 % for healthy donor T cells, Fig. 2b, c). This was accompanied by considerably lower concentrations of IFN-? in cell culture
supernatants (47.7 vs. 2100.9 pg/ml for healthy donor T cells, Fig. 2d). These results recapitulate the clinical experience with an inability of the patients’
T cells to perform blinatumomab-mediated lysis of ALL cells.