Poster Session B   |   7:00am Expo - Hall A & C   |   Poster ID #114

Development of an immunoPET imaging method for chimeric antigen receptor (CAR) T-cell therapies

Academic Research
Prevention, Early Detection, Implementation, and Dissemination
FDA Status:
Not Applicable
CPRIT Grant:
Cancer Site(s):
Shelby Harris
The University of Texas Southwestern Medical Center
Aditi Mulgaonkar
The University of Texas Southwestern Medical Center
Yaxing Yang
The University of Texas Southwestern Medical Center
Orhan Oz
The University of Texas Southwestern Medical Center
Praveen Ramakrishnan Geethakumari
The University of Texas Southwestern Medical Center
Xiankai Sun
The University of Texas Southwestern Medical Center


Chimeric antigen receptor (CAR) T-cell therapies have revolutionized treatment of B-cell hematological malignancies, with complete remission in ~70-90% of the cases. However, ~50% of these cancers relapse within the first year post-therapy. Cancer resistance or relapse with the loss of cluster of differentiation 19 (CD19) antigen is a common challenge encountered with CD19 directed CAR T-cell therapies when treating advanced B-cell malignancies. Thus, a noninvasive real-time imaging approach to assess the dynamic expression of CD19 antigen is highly desirable to ensure precision treatment. In this preliminary work, we tested the feasibility of an immuno–positron emission tomography (ImmunoPET) imaging approach using an anti-CD19 monoclonal antibody, tafasitamab (TAF) radiolabeled with zirconium-89 ([89Zr]Zr-DFO-TAF) to detect dynamic variation in CD19 expression. 


TAF was conjugated with p-isothiocyanatobenzyl-deferoxamine chelator (p-NCS-Bz-DFO) followed by radiolabeling with 89Zr to yield the radiotracer ([89Zr]Zr-DFO-TAF). The characterization of [89Zr]Zr-DFO-TAF was performed by fast protein liquid chromatography equipped with a radio-detector (radio-FPLC) to measure its radiochemical purity and molar activity as well as a Lindmo assay to assess its immunoreactivity using Raji (CD19+) cells. The in vivo CD19 targeting specificity of [89Zr]Zr-DFO-TAF was evaluated in immunocompromised (NOD/SCID) and immunocompetent (C57BL/6) mouse strains bearing either CD19+ (Raji) or CD19low/none (K562-s; MC38 as non-B-cell malignancy control) subcutaneous tumors. After intravenous injection of the radiotracer (~35-50 µCi), the mice were scanned over a period of six days. Quantitative PET analysis in regions of interest was performed after co-registration with the corresponding computed tomography (CT) images. Immediately after imaging, the mice were sacrificed and tumors were processed for anti-CD19 immunohistochemistry (IHC) staining.


[89Zr]Zr-DFO-TAF was synthesized with >90% radiochemical purity and 1.5-4 mCi/mg molar activity. Its immunoreactivity was 100 ± 10% (n = 3). Surprisingly, the comparative imaging studies in NOD/SCID mice bearing Raji (CD19+) versus MC38 and K562-s (CD19low/none) tumors showed no differences in both, tumor uptake values (~3.3% injected dose per gram (ID/g) versus ~4% ID/g and 3.3% ID/g, respectively) and tumor-to-muscle background contrast ratios (T/M) (~4.4 versus ~4.6 and 4.3, respectively). Of note, the CD19 expression levels in the tumors were confirmed by IHC staining. Further, a comparative study was conducted in two mouse strains (NOD/SCID versus C57BL/6) bearing the same MC38 tumors. It showed significantly higher tumor uptake (~7.4% ID/g versus ~4% ID/g; p = 0.018) and T/M ratios (~11.4 versus ~4.6; p = 0.001) in C57BL/6 mice than in the NOD/SCID mice. Further examination of the data and TAF’s binding mechanism revealed that the enhanced fragment crystallizable (Fc)-receptor interactions of [89Zr]Zr-DFO-TAF resulting from TAF’s Fc domain mutations might predominate over the anticipated CD19-specific uptake in the tumors.


Introducing two Fc-domain mutations (S239D and I332E) in the structure of TAF has demonstrated enhanced effector function due to increased Fc-receptor interactions, thereby improving its therapeutic potential. However, these non-specific Fc interactions impair its potential for immunoPET imaging of CD19. Therefore, we will explore the use of an Fc-silenced anti-CD19 antibody instead.