Frequency and Clinical Characteristics Associated with Putative CAR Targets ADGRE2, CCR1, CD70, and LILRB2 in Acute Myeloid Leukemia
Authors: Shahswar Rabia , Kloos Arnold , Gabdoulline Razif , Mohanty Sagarajit , Koenecke Christian , Wichmann Martin , Neziri Blerina , Klement Piroska , Schiller Johannes , Klesse Sabrina , Liebich Alessandro , Lai Courteney , Krauter Jürgen , Trummer Arne , Fiedler Walter , Kirchner Hartmut , Heil Gerhard , Li Zhixiong , Hambach Lothar , Ganser Arnold , Thol Felicitas , Heuser Michael 
Published: October 2019

Abstract
Background: ADGRE2, CCR1, CD70, and LILRB2 expressed on the surface of myeloid blasts but not normal hematopoietic stem cells, T cells or other tissues have been recently suggested as candidate chimeric antigen receptor (CAR) targets for engineered T cells in acute myeloid leukemia (AML) patients.
Aim: To validate the expression pattern of the recently identified candidate CAR targets ADGRE2, CCR1, CD70, and LILRB2 on leukemic blasts in a cohort of newly diagnosed AML patients.
Methods: 109 patients with de novo (n=87) or secondary (n=22) AML were included in the analysis. Patients were classified according to the 2008 WHO classification and cytogenetically characterized by chromosome banding analysis. Molecular analyses were performed by Sanger and next-generation sequencing (NGS) with a panel of 46 genes. Bone marrow or peripheral blood samples from the time of first diagnosis were obtained to perform multi-color flow cytometry analysis evaluating the expression levels of ADGRE2 (also known as EMR2 or CD132), CCR1 (also known as CD191), CD70 and LILRB2 (also known as CD85d) antigens on the surface of myeloid blasts. Patients with expression of the marker on ≥20% of blast cells were defined positive. Informed consent was obtained from all patients in accordance to the declaration of Helsinki and institutional guidelines.
Results: ADGRE2, CCR1, CD70 and LILRB2 were expressed in 100%, 70%, 27.5%, 27.5% of patients with a median expression on myeloid blasts of 87.8% (range 30.4-99.8%), 43.9% (range 21.1-86.2%), 29.0% (range 20.0-55.4%), and 35.6% (range 20.7-90.6%), respectively. A subset analysis was performed to determine expression levels of the candidate targets in CD3 positive cells. Of patients with positive marker expression on blast cells ADGRE2, CCR1, CD70 and LILRB2 were expressed on 14.5% (range 0.0-64.2%), 19.9% (0.0-73.0%), 15.3% (range 0.0-35.1%), and 2% (range 0.1-18.4%) of CD3+ T cells, respectively. The proportion of patients with ≥80% expression on blasts was 61.5% (n=67), 0.9% (n=1) and 3.7% (n=4) for ADGRE2, CCR1 and LILRB2, respectively. Of those 53.7%, 0%, and 100% had marker expression <20% on CD3+ T cells and 15%, 0%, and 25% had no expression (<1%) of ADGRE2, CCR1 and LILRB2 on CD3+ T cells.
There were no significant differences in the distribution of age, type of AML, sex, WHO subtypes, cytogenetic risk according to 2017 ELN classification, WBC count, hemoglobin and platelet count or for the type of consolidation treatment between CCR1, CD70, and LILRB2 positive compared to negative patients except for a higher platelet count in LILRB2 positive patients (p=0.009). To evaluate the association between mean expression level and mutational profile molecular analyses were performed as mentioned above and correlated with expression levels. More blast cells expressed CCR1 in CEBPA mutated patients compared to CEBPA wildtype patients (43.7% vs. 32.8%, p=0.003), while fewer blast cells expressed LILRB2 in IDH2 mutated compared to wildtype patients (9% vs. 16.8%, p=0.027). However, no other mutations correlated with the expression of the candidate CAR targets. Next, Pearson correlation was calculated to determine co-expression of the candidate CAR targets. A positive correlation was found between CCR1 and CD70 expression on blast cells (R=0.673, p<0.001). ADGRE2 correlated with LILRB2 and CD70 expression on CD3+ T cells (R=0.631, p<0.001 and R=0.416, p<0.001, respectively). CCR1 correlated with CD70 and LILRB2 on CD3+ T cells (R=0.807, p<0.001 and R=0.46, p<0.001, respectively). The rate of complete remission was similar for patients with and without expression of CCR1, CD70 and LILRB2 (82.2% vs. 14.5%, p=0.115; 73.3% vs. 20%, p=0.349; 83.3% vs. 13.3%, p=0.828, respectively). Overall survival, event-free and relapse-free survival were also similar for patients with and without expression of CCR1, CD70 and LILRB2.
Conclusions: Our data show variable expression levels of candidate CAR targets in AML blast cells with ADGRE2 being expressed at high levels in all patients. However, expression levels were not specifically associated with patient characteristics or outcome. Our findings favor ADGRE2 as potentially suitable for CAR targeting as it had the most favorable expression profile on blasts and T cells in AML patients.

Disclosures
Koenecke: BMS: Consultancy; Amgen: Consultancy; abbvie: Consultancy; Roche: Consultancy. Fiedler:Daiichi Sankyo: Other: support for meeting attendance; Gilead: Other: support for meeting attendance; Amgen: Other: support for meetíng attendance; Pfizer: Research Funding; Amgen: Research Funding; Amgen: Patents & Royalties; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; ARIAD/Incyte: Membership on an entity's Board of Directors or advisory committees, support for meeting attendance; Amgen: Consultancy, Membership on an entity's Board of Directors or advisory committees; GSO: Other: support for meeting attendance; Teva: Other: support for meeting attendance; JAZZ Pharmaceuticals: Other: support for meeting attendance. Ganser:Novartis: Membership on an entity's Board of Directors or advisory committees.