Open Access

Increase of PD-L1 expressing B-precursor ALL cells in a patient resistant to the CD19/CD3-bispecific T cell engager antibody blinatumomab

  • Thomas Köhnke1, 2,
  • Christina Krupka1, 2,
  • Johanna Tischer1,
  • Thomas Knösel3 and
  • Marion Subklewe1, 2Email author
Journal of Hematology & Oncology20158:111

https://doi.org/10.1186/s13045-015-0213-6

Received: 12 August 2015

Accepted: 28 September 2015

Published: 8 October 2015

Abstract

The bispecific T cell engager blinatumomab has shown encouraging clinical activity in B-precursor acute lymphoblastic leukemia (ALL). However, about half of relapsed/refractory patients do not respond to therapy. Here, we present the case of a 32-year-old male patient with refractory B-precursor ALL who was resistant to treatment with blinatumomab. Bone marrow immunohistochemistry revealed T cell infiltrates and an increase in programmed death-ligand 1 (PD-L1)-positive ALL cells as a potential immune escape mechanism. We were able to recapitulate the clinical observation in vitro by showing that blinatumomab was not able to mediate cytotoxicity of CD19-positive ALL cells using autologous T cells. In contrast, the addition of healthy donor T cells led to lysis of ALL cells.

These results strongly encourage further systematic evaluation of checkpoint molecules in cases of blinatumomab treatment failure and might highlight a possible mechanism to overcome resistance to this otherwise highly effective treatment.

Keywords

ALLImmunotherapyBlinatumomabImmune checkpointsT cellsCombination therapy

Background

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 μg/day for 7 days and 28 μg/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/μl on day 0 vs. 217/μl on day 28, Fig. 1b) including decreased T cells (960 CD3-positive T cells/μl on day 5 vs. 180/μl 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/μl on day 0, 986/μl on day 7, 464/μl on day 14, 368/μl on day 21, and 217/μl 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-γ 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-γ production. Cell cultures with control-Bite® did not show any IFN-γ production

Finally, we collected the patient’s peripheral blood after blinatumomab treatment and purified CD3-positive T cells (EasySep™ Human CD3 Positive Selection Kit II, Stemcell Technologies, Vancouver, British Columbia, Canada). Of either patient or healthy donor CD3-positive T cells, 2.5 × 105 were cocultured with 5 × 105 of the patient’s ALL cells (CD3-negative cells) with blinatumomab or control-BiTE® at a dose of 5 ng/ml on 3 × 104 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-γ (IFN-γ) concentration in cell culture supernatants was measured by cytometric bead array (BD™ CBA Human IFN-γ 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-γ 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.

Discussion and conclusions

Taken together, our data suggests a role of PD-L1 in treatment resistance to blinatumomab in our patient. This is, to our knowledge, the first report of increased PD-L1 positivity in a patient receiving blinatumomab and should encourage a systematic evaluation of the relevance of this resistance mechanism in patients receiving bispecific T cell engager therapy. A large body of evidence has been suggesting a key role of the PD-1/PD-L1 axis in attenuating anti-tumor immune responses [16, 17]. Recently, we described this mechanism of resistance in the context of BiTE® antibody immunotherapy in vitro [18]. Detecting an increase of PD-L1 in a patient receiving blinatumomab therefore highlights the relevance of this mechanism in vivo.

Additionally, several resistance mechanisms to antibody-based immunotherapy have been reported including variation of target antigen expression (which might be present initially or develop during therapy), activation of alternative signaling pathways, and anti-antibody formation [1922]. In the context of novel immunotherapies, including blinatumomab, assessment of resistance mechanisms is limited and warrants further investigation. Upregulation of checkpoint molecules represents an adaptive resistance to anti-tumor immunity which we hypothesize will also take place in other T cell recruiting antibody formats (e.g., DARTS, diabodies) as well as adoptive T cell therapies (e.g., CAR T cells, tumor antigen-specific TCR T cells). It will be important to conduct close monitoring of biomarkers and careful consideration of specific time points recognizing the dynamic interplay of receptor-ligand interactions. Further studies are warranted to analyze the significance of the PD-1/PD-L1 interplay as a resistance mechanism to blinatumomab. Ultimately, combinatorial approaches might have the potential to revert T cell-induced immune escape strategies and avoid treatment failure in these otherwise highly effective treatments.

Statement of informed consent

Written informed consent was obtained from the patient for publication of this manuscript. A copy of the written consent is available for review by the Editor-in-Chief of this journal.

Abbreviations

ALL: 

acute lymphoblastic leukemia

CAR T cells: 

chimeric antigen receptor T cells

DARTs: 

dual affinity re-targeting molecules

GMALL: 

German multicenter study group on adult acute lymphoblastic leukemia

IFN-γ: 

interferon-γ

PD-1: 

programmed cell death-1

PD-L1: 

programmed death-ligand 1

TCR: 

T cell receptor

Declarations

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
Department of Internal Medicine III, Ludwig-Maximilians-Universität (LMU)
(2)
Clinical Cooperation Group Immunotherapy at the Helmholtz Zentrum München
(3)
Institute of Pathology, Ludwig-Maximilians-Universität (LMU)

References

  1. Suresh T, Lee LX, Joshi J, Barta SK. New antibody approaches to lymphoma therapy. J Hematol Oncol. 2014;7(1):58.PubMed CentralView ArticlePubMedGoogle Scholar
  2. Wu J, Fu J, Zhang M, Liu D. Blinatumomab: a bispecific T cell engager (BiTE) antibody against CD19/CD3 for refractory acute lymphoid leukemia. J Hematol Oncol. 2015;8(1):104.PubMed CentralView ArticlePubMedGoogle Scholar
  3. Zugmaier G, Klinger M, Schmidt M, Subklewe M. Clinical overview of anti-CD19 BiTE(®) and ex vivo data from anti-CD33 BiTE(®) as examples for retargeting T cells in hematologic malignancies. Mol Immunol. 2015;67(2 Pt A):58–66.View ArticlePubMedGoogle Scholar
  4. Topp MS, Gökbuget N, Stein AS, Zugmaier G, O'Brien S, Bargou RC, et al. Safety and activity of blinatumomab for adult patients with relapsed or refractory B-precursor acute lymphoblastic leukaemia: a multicentre, single-arm, phase 2 study. Lancet Oncol. 2014;16(1):57–66.View ArticlePubMedGoogle Scholar
  5. Portell CA, Wenzell CM, Advani AS. Clinical and pharmacologic aspects of blinatumomab in the treatment of B-cell acute lymphoblastic leukemia. Clin Pharmacol. 2013;5 Suppl 1:5–11.PubMed CentralPubMedGoogle Scholar
  6. Bassan R. Toward victory in adult ALL: blinatumomab joins in. Blood. 2012;120(26):5094–5.View ArticlePubMedGoogle Scholar
  7. Eyre TA, Collins GP. Immune checkpoint inhibition in lymphoid disease. Br J Haematol. 2015;170(3):291–304.View ArticlePubMedGoogle Scholar
  8. Shi L, Chen S, Yang L, Li Y. The role of PD-1 and PD-L1 in T-cell immune suppression in patients with hematological malignancies. J Hematol Oncol. 2013;6(1):74.PubMed CentralView ArticlePubMedGoogle Scholar
  9. Ohaegbulam KC, Assal A, Lazar-Molnar E, Yao Y, Zang X. Human cancer immunotherapy with antibodies to the PD-1 and PD-L1 pathway. Trends Mol Med. 2015;21(1):24–33.View ArticlePubMedGoogle Scholar
  10. Homet Moreno B, Ribas A. Anti-programmed cell death protein-1/ligand-1 therapy in different cancers. Br J Cancer. 2015;112(9):1421–7.PubMed CentralView ArticlePubMedGoogle Scholar
  11. Ramsay AG. Immune checkpoint blockade immunotherapy to activate anti-tumour T-cell immunity. Br J Haematol. 2013;162(3):313–25.View ArticlePubMedGoogle Scholar
  12. Brahmer JR, Tykodi SS, Chow LQM, Hwu W-J, Topalian SL, Hwu P, et al. Safety and activity of anti-PD-L1 antibody in patients with advanced cancer. N Engl J Med. 2012;366(26):2455–65.PubMed CentralView ArticlePubMedGoogle Scholar
  13. Kefford R, Ribas A, Hamid O, Robert C, Daud A, Wolchok JD, et al. Clinical efficacy and correlation with tumor PD-L1 expression in patients (pts) with melanoma (MEL) treated with the anti-PD-1 monoclonal antibody MK-3475. ASCO Meet Abstr. 2014;32(15_suppl):3005. May 20.Google Scholar
  14. Smith AD, Roda D, Yap TA. Strategies for modern biomarker and drug development in oncology. J Hematol Oncol. 2014;7(1):70.PubMed CentralView ArticlePubMedGoogle Scholar
  15. Krupka C, Kufer P, Kischel R, Zugmaier G, Bogeholz J, Köhnke T, et al. CD33 target validation and sustained depletion of AML blasts in long-term cultures by the bispecific T-cell-engaging antibody AMG 330. Blood. 2014;123(3):356–65.View ArticlePubMedGoogle Scholar
  16. Madorsky Rowdo FP, Baron A, Urrutia M, Mordoh J. Immunotherapy in cancer: a combat between tumors and the immune system; you win some, you lose some. Front Immunol. 2015;26:6.Google Scholar
  17. Taube JM, Anders RA, Young GD, Xu H, Sharma R, McMiller TL, et al. Colocalization of inflammatory response with B7-H1 expression in human melanocytic lesions supports an adaptive resistance mechanism of immune escape. Sci Transl Med Am Assoc Adv Sci. 2012;4(127):127ra37–7.Google Scholar
  18. Krupka C, Kufer P, Kischel R, Zugmaier G, Lichtenegger FS, Köhnke T, et al. Blockade of the PD-1/PD-L1 axis augments lysis of AML cells by the CD33/CD3-BiTE® antibody construct AMG 330: reversing a T-cell induced immune escape mechanism. Leukemia: Nature Publishing Group; 2015.Google Scholar
  19. Press OW, Leonard JP, Coiffier B, Levy R, Timmerman J. Immunotherapy of non-Hodgkin’s lymphomas. Hematol Am Soc. 2001;2001(1):221–40.View ArticleGoogle Scholar
  20. Shuptrine CW, Surana R, Weiner LM. Monoclonal antibodies for the treatment of cancer. Semin Cancer Biol. 2012;22(1):3–13.PubMed CentralView ArticlePubMedGoogle Scholar
  21. Scott AM, Wolchok JD, Old LJ. Antibody therapy of cancer. Nat Publ Group. 2012;12(4):278–87.Google Scholar
  22. Reslan L, Dalle S, Dumontet C. Understanding and circumventing resistance to anticancer monoclonal antibodies. MAbs Landes Bioscience; 2009;1(3):222–9.Google Scholar

Copyright

© Köhnke et al. 2015

Advertisement