Research Grant Update from Memorial Sloan Kettering.
TeamConnor is pleased to share the most recent research update from one of our past childhood cancer research grant recipients.
Simone Krebs, MD, MS is the Assistant Attending, Molecular Imaging and Therapy Service as well as the Assistant Professor of Radiology, Weill Cornell Medicine at Memorial Sloan Kettering Cancer Center in New York.
Thank you, Dr. Krebs and your team for the important and needed research that you all are overseeing for malignant childhood gliomas.
“Novel molecular imaging tools for monitoring CAR T cells in malignant childhood gliomas”
We have established an orthotopic DIPG mouse model using a patient-derived cell line. However, due to the markedly slow growth of these tumor cells, it required more than five months after engraftment into the mouse brain until a tumor was visualized. To overcome this limitation, we have purchased a new glioma cell line bearing the H3K27M mutation, similar to other DIPG tumors. This cell line is also marked with luciferase to facilitate monitoring of tumor growth using bioluminescence imaging. We observed no intracranial tumor growth (cell doses up to 200k per mouse) over a period of 7 months (inoculated a total of 20 mice). Following s.c. engraft of 4.5x10e6 glioma cells/mouse (n=3 mice) initial weak bioluminescence signal was observed that spontaneously resolved without treatment. Due to the challenges in establishing glioma PDX models, we are now pursuing in vivo treatments with GD2+ LAN-1 cells provided by our collaborator.
In the meantime, we have made progress with our novel imaging tools including 89Zr-labeled antibodies targeting activation markers and a humanized anti-DOTA scFv reporter platform to facilitate clinical translation.
Figure 1. Patient-derived H3-K27M+ DIPG line highly expresses GD2.
Expression of GD2 was confirmed using flow cytometry. (Blue: control, Red: cells stained with antibody against GD2). (Shift to the right confirms positivity for GD2 expression)
Figure 2. In vitro cytotoxicity assay shows that GD2 CAR T cells specifically kill GD2-expressing DIPG cell line (patient-derived; red line) and GD2-expressing neuroblastoma (LAN-1; green line). In contrast, a GD2-negative cell line is not killed (black line). Representative result is shown.
Figure 3. MRI images of two representative mice at 2 months post-tumor inoculation into the brain stem. (Patient-derived DIPG mouse model)
A, B) Mouse M#1: Axial slices of T2- weighted images show low signal abnormality, possibly indicating tumor (area circled in red font). Two consecutive slices are shown.
C, D) Mouse M#2: Axial slices of T2-weighted images show high signal abnormality possibly indicating tumor (area circled in red font). Two consecutive slides are shown.
Figure 4. MRI images of two representative mice at 5 months post-tumor inoculation into the brain stem. (Patient-derived DIPG)
A, B) Mouse M#1
Axial slices of T2- weighted images show a large high signal abnormality in the brain compatible with tumor (area circled in red font). Two consecutive slides are shown.
C, D) Mouse M#2
Axial slices of T2-weighted images show a vague high signal abnormality possibly indicating tumor (area circled in red font). Two consecutive slides are shown.
Figure 5. Radiochemical purity of 89Zr-AbGITR and 89Zr-AbOX40 as-evaluated by instant thin-layer chromatography (post-purification). This confirms successful radiolabeling and purity of the radiolabeled antibodies targeting GITR and OX40.
Figure 6. DFO-conjugates of the anti-GITR and anti-OX40 antibodies show retained immunoreactivity for their cognate antigens. Sensorgrams showing robust dose response curves and kinetic constants (ka, kd, KD and Rmax) for (A) anti-GITR and (C) anti-OX40 parent antibodies and their DFO-conjugated variants (B, D).
This analysis confirms that the modification of the antibodies through (radio)labeling does not alter in the binding profile of the GITR and OX40 antibodies.
Figure 7. 89Zr-AbGITR and 89Zr-AbOX40 PET/CT characterize activation state of tumor-infiltrating lymphocytes in a syngeneic mouse model of melanoma. (A) MIP PET/CT images (top), coronal (middle), and axial PET images (bottom) at 48 h p.i. demonstrate intratumoral T-cell accumulation and expression of activation markers (white arrows). On 89Zr-AbGITR PET images using a total injected protein mass of 5 µg, uptake is also seen in lymph nodes including in tumor-draining lymph node (right inguinal node, orange arrow) and spleen, confirming additional sites of T-cell localization. Minimal uptake above background at the tumor site is noted on IgG images. (B) Image-based biodistribution. TNR: tumor-to-normal tissue ratio; B: blood; L: liver; K: kidney; M: muscle. (n = 3); *, P < 0.05; **, P < 0.01.