Axl-targeted cancer imaging with humanized antibody h173
Dan Li1, Shuanglong Liu, Ren Liu
1Molecular Imaging Center, Department of Radiology, University of Southern California, 2250 Alcazar St. CSC103, Los Angeles, CA, 90033, USA.
Purpose:
The tyrosine kinase receptor Axl is overexpressed in various types of cancer and correlated with cancer malignancy. Selective Axl blockade reduces tumor growth and metastasis. The purpose of this study was to examine whether the humanized anti-Axl antibody humanized 173 (h173) labeled with near-infrared fluorescence (NIRF) dye Cy5.5 could be applied as a molecular imaging probe for NIRF imaging of Axl expression in tumor models.
Procedures:
NIRF dye Cy5.5 was conjugated to h173 or human normal immunoglobulin G (hIgG) control through amino groups. The resulting probes were evaluated in both A549 (Axl positive) and NCI-H249 (Axl negative) lung cancer xenografts through in vivo NIRF imaging. Ex vivo imaging and probe distribution assay were also carried out to confirm the in vivo imaging results.
Results:
After conjugation, binding activity of h173-Cy5.5 was determined to be 97.75 % ± 2.09 % of the unmodified h173. In vitro fluorescence-activated cell sorting (FACS) and fluorescence microscopy analysis validated the specific binding of h173 toward Axl-positive A549 cells. h173-Cy5.5 was then applied to image Axl expression in vivo. In A549 (Axl positive) cancer xenografts, the tumor uptake of h173-Cy5.5 was significantly higher than that of the hIgG-Cy5.5 control (P < 0.05) at late time points (1, 2, 3, 4, and 7 days). On the contrary, in NCI-H249 (Axl negative) cancer xenografts, the tumor uptake of both hIgG-Cy5.5 and h173-Cy5.5 was low and showed no significant difference (P > 0.05) at all time points examined. Ex vivo imaging and immunofluorescence staining analysis further validated the in vivo imaging results.
Conclusions:
Collectively, all in vitro, in vivo, and ex vivo data suggested that h173-Cy5.5 could serve as a valid probe for Axl-targeted cancer imaging, which could therefore aid in tumor diagnosis, prognosis, and treatment monitoring.
Insights
A novel near-infrared fluorescence (NIRF) imaging probe, humanized 173 (h173)-Cy5.5, effectively targets and visualizes Axl-expressing tumors. This advancement holds promise for improved cancer diagnosis and monitoring.
Area of Science:
- Oncology
- Molecular Imaging
- Bioconjugation
Background:
- Axl receptor tyrosine kinase is frequently overexpressed in cancers, correlating with malignancy and metastasis.
- Targeting Axl offers a therapeutic strategy to reduce tumor growth and spread.
Purpose of the Study:
- To evaluate the humanized anti-Axl antibody humanized 173 (h173) conjugated with near-infrared fluorescence (NIRF) dye Cy5.5 as a molecular imaging probe.
- To assess the probe's efficacy in NIRF imaging of Axl expression in preclinical tumor models.
Main Methods:
- Conjugation of Cy5.5 dye to h173 antibody and a control human IgG (hIgG).
- In vivo NIRF imaging of Axl-positive (A549) and Axl-negative (NCI-H249) lung cancer xenografts.
- Ex vivo imaging and probe distribution assays to validate in vivo findings.
Main Results:
- h173-Cy5.5 demonstrated specific binding to Axl-positive cells in vitro.
- Significantly higher tumor uptake of h173-Cy5.5 was observed in Axl-positive xenografts compared to hIgG-Cy5.5 controls.
- No significant difference in probe uptake was found in Axl-negative xenografts, confirming probe specificity.
Conclusions:
- The h173-Cy5.5 probe is a valid tool for Axl-targeted cancer imaging.
- This probe can potentially aid in tumor diagnosis, prognosis, and treatment monitoring.
