Imaging Activated T Cells Predicts Response to Cancer Vaccines



Israt S. Alam1,2, Aaron T. Mayer1,2,3, Idit Sagiv-Barfi4, Kezheng Wang1,5, Ophir Vermesh1,2, Debra K. Czerwinski4, Emily M. Johnson1,2,6, Michelle L. James1,2,6, Ronald Levy4, and Sanjiv S. Gambhir1,2


  1. Department of Radiology
  2. Molecular Imaging Program at Stanford
  3. Department of Bioengineering, and
  4. Division of Oncology, Department of Medicine, Stanford University, Stanford, California, USA.
  5. Department of Radiology, The Fourth Hospital of Harbin Medical University and Molecular Imaging Center of Harbin Medical University, Harbin, China.
  6. Department of Neurology and Neurological Sciences, Stanford University, Stanford, California, USA.


In situ cancer vaccines are under active clinical investigation, given their reported ability to eradicate both local and disseminated malignancies. Intratumoral vaccine administration is thought to activate a T cell–mediated immune response, which begins in the treated tumor and cascades systemically. In this study, we describe a PET tracer (64Cu-DOTA-AbOX40) that enabled noninvasive and longitudinal imaging of OX40, a cell-surface marker of T cell activation. We report the spatiotemporal dynamics of T cell activation following in situ vaccination with CpG oligodeoxynucleotide in a dual tumor–bearing mouse model. We demonstrate that OX40 imaging was able to predict tumor responses on day 9 after treatment on the basis of tumor tracer uptake on day 2, with greater accuracy than both anatomical and blood-based measurements. These studies provide key insights into global T cell activation following local CpG treatment and indicate that 64Cu-DOTA-AbOX40 is a promising candidate for monitoring clinical cancer immunotherapy strategies.

CovalX Technology Used (Click each option to learn more)



OX40-DOTA conjugation with the DOTA-NHS ester was performed by optimizing the DOTA conjugation with murine OX40 mAb resuspended in PBS at 0.5 mg/ml. Then, the conjugation was buffer exchanged into metal-free HEPES buffer with a pH of 8.8 and incubated at 4 °C overnight before being quenched with Tris buffer, buffer exchanged again into metal-free water and then concentrated. An aliquot of this solution and an unconjugated sample of OX40 Ab were used for MALDI-TOF mass spectrometry. The DOTA/mAb ratio was calculated by looking at the change in mass seen during the mass spectrometry with a MALDI TOF mass spectrometer that had been modified with a CovalX high mass detection system and then dividing the mass by the mass of a single DOTA substituent. The use of mass spectrometry analysis allowed researchers to determine the average number of chelates per Ab molecules.


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