Molecular Imaging and Biology

, Volume 19, Issue 6, pp 903–914 | Cite as

Development of Novel ImmunoPET Tracers to Image Human PD-1 Checkpoint Expression on Tumor-Infiltrating Lymphocytes in a Humanized Mouse Model

  • Arutselvan Natarajan
  • Aaron T. Mayer
  • Robert E. Reeves
  • Claude M. Nagamine
  • Sanjiv Sam Gambhir
Research Article



It is well known that cancers exploit immune checkpoints (programmed death 1 receptor (PD-1) and its ligand (PD-L1)) to evade anti-tumor immune responses. Although immune checkpoint (IC) blockade is a promising approach, not all patients respond. Hence, imaging of tumor-infiltrating lymphocytes (TILs) is of high specific interest, as they are known to express PD-1 during activation and subsequent exhaustion in the tumor microenvironment and are thought to be potentially predictive of therapeutic responses to IC blockade.


We developed immune-tracers for positron emission tomography (PET) to image hPD-1 status of human peripheral blood mononuclear cells (hPBMCs) adoptively transferred to NOD-scid IL-2Rγnull (NSG) mice (hNSG) bearing A375 human skin melanoma tumors. The anti-PD-1 human antibody (IgG; keytruda) was labeled with either Zr-89 or Cu-64 radiometals to image PD-1-expressing human TILs in vivo.


[89Zr] Keytruda (groups = 2; NSG-ctl (control) and hNSG-nblk (non-blocking), n = 3–5, 3.2 ± 0.4 MBq/15–16 μg/200 μl) and [64Cu] Keytruda (groups = 3; NSG-ctl, NSG-blk (blocking), and hNSG-nblk; n = 4, 7.4 ± 0.4 MBq /20-25 μg/200 μl) were administered in mice. PET-CT scans were performed over 1–144 h ([89Zr] Keytruda) and 1–48 h ([64Cu] Keytruda) on mice. hNSG mice exhibited a high tracer uptake in the spleen, lymphoid organs and tumors. At 24 h, human TILs homing into melanoma of hNSG-nblk mice exhibited high signal (mean %ID/g ± SD) of 3.8 ± 0.4 ([89Zr] Keytruda), and 6.4 ± 0.7 ([64Cu] Keytruda), which was 1.5- and 3-fold higher uptake compared to NSG-ctl mice (p = 0.01), respectively. Biodistribution measurements of hNSG-nblk mice performed at 144 h ([89Zr] Keytruda) and 48 h ([64Cu] Keytruda) p.i. revealed tumor to muscle ratios as high as 45- and 12-fold, respectively.


Our immunoPET study clearly demonstrates specific imaging of human PD-1-expressing TILs within the tumor and lymphoid tissues. This suggests these anti-human-PD-1 tracers could be clinically translatable to monitor cancer treatment response to IC blockade therapy.

Key words

ImmunoPET Tumor-infiltrating lymphocytes PD-1 Keytruda 64-Cu 89-Zr 



We would like to thank The Canary Foundation, The Ben and Catherine Ivy Foundation, and the National Cancer Institute for their support and for helping to fund this research. We acknowledge the supports of Drs. Mark Stolowitz, Timothy Doyle, Frezghi Habte, and Lingyun Xu; Sindhuja Ramakrishnan; and Michelle Tran for the experiments performed. MicroPET/CT imaging and gamma counter measurements were performed in the SCi3 Stanford Small Animal Imaging Service Center.

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflict of interest.

Supplementary material

11307_2017_1060_MOESM1_ESM.docx (3.1 mb)
ESM 1(PDF 3.07 mb)


  1. 1.
    Gentzler R, Hall R, Kunk PR et al (2016) Beyond melanoma: inhibiting the PD-1/PD-L1 pathway in solid tumors. Immunotherapy 8:583–600CrossRefPubMedGoogle Scholar
  2. 2.
    Sharma P, Allison JP (2015) The future of immune checkpoint therapy. Science 348:56–61CrossRefPubMedGoogle Scholar
  3. 3.
    Topalian SL, Hodi FS, Brahmer JR et al (2012) Safety, activity, and immune correlates of anti-PD-1 antibody in cancer. N Engl J Med 366:2443–2454CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Hodi FS, O’Day SJ, McDermott DF et al (2010) Improved survival with ipilimumab in patients with metastatic melanoma. N Engl J Med 363:711–723CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Robert C, Thomas L, Bondarenko I et al (2011) Ipilimumab plus dacarbazine for previously untreated metastatic melanoma. N Engl J Med 364:2517–2526CrossRefPubMedGoogle Scholar
  6. 6.
    Yun S, Vincelette ND, Green MR et al (2016) Targeting immune checkpoints in unresectable metastatic cutaneous melanoma: a systematic review and meta-analysis of anti-CTLA-4 and anti-PD-1 agents trials. Cancer Med 5:1481–1491CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Wolchok JD, Kluger H, Callahan MK et al (2013) Nivolumab plus ipilimumab in advanced melanoma. N Engl J Med 369:122–133CrossRefPubMedGoogle Scholar
  8. 8.
    Berghoff AS, Ricken G, Widhalm G et al (2014) PD1 (CD279) and PD-L1 (CD274, B7H1) expression in primary central nervous system lymphomas (PCNSL). Clin Neuropathol 33:42–49CrossRefPubMedGoogle Scholar
  9. 9.
    Brown JA, Dorfman DM, Ma FR et al (2003) Blockade of programmed death-1 ligands on dendritic cells enhances T cell activation and cytokine production. J Immunol 170:1257–1266CrossRefPubMedGoogle Scholar
  10. 10.
    Franceschini D, Paroli M, Francavilla V et al (2009) PD-L1 negatively regulates CD4 + CD25 + Foxp3+ Tregs by limiting STAT-5 phosphorylation in patients chronically infected with HCV. J Clin Invest 119:551–564CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Hamid O, Robert C, Daud A et al (2013) Safety and tumor responses with lambrolizumab (anti-PD-1) in melanoma. N Engl J Med 369:134–144CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Mamalis A, Garcha M, Jagdeo J (2014) Targeting the PD-1 pathway: a promising future for the treatment of melanoma. Arch Dermatol Res 306:511–519CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Drake CG, Jaffee E, Pardoll DM (2006) Mechanisms of immune evasion by tumors. Adv Immunol 90:51–81CrossRefPubMedGoogle Scholar
  14. 14.
    Fridman WH, Pages F, Sautes-Fridman C et al (2012) The immune contexture in human tumours: impact on clinical outcome. Nat Rev Cancer 12:298–306CrossRefPubMedGoogle Scholar
  15. 15.
    Mellman I, Coukos G, Dranoff G (2011) Cancer immunotherapy comes of age. Nature 480:480–489CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Agata Y, Kawasaki A, Nishimura H et al (1996) Expression of the PD-1 antigen on the surface of stimulated mouse T and B lymphocytes. Int Immunol 8:765–772CrossRefPubMedGoogle Scholar
  17. 17.
    Mullard A (2013) New checkpoint inhibitors ride the immunotherapy tsunami. Nat Rev Drug Discov 12:489–492CrossRefPubMedGoogle Scholar
  18. 18.
    Zou W, Chen L (2008) Inhibitory B7-family molecules in the tumour microenvironment. Nat Rev Immunol 8:467–477CrossRefPubMedGoogle Scholar
  19. 19.
    Rosenberg SA (2004) Development of effective immunotherapy for the treatment of patients with cancer. J Am Coll Surg 198:685–696CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Ohaegbulam KC, Assal A, Lazar-Molnar E et al (2015) Human cancer immunotherapy with antibodies to the PD-1 and PD-L1 pathway. Trends Mol Med 21:24–33CrossRefPubMedGoogle Scholar
  21. 21.
    Topalian SL, Sznol M, McDermott DF et al (2014) Survival, durable tumor remission, and long-term safety in patients with advanced melanoma receiving nivolumab. J Clin Oncol 32:1020–1030CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Robert C, Ribas A, Wolchok JD et al (2014) Anti-programmed-death-receptor-1 treatment with pembrolizumab in ipilimumab-refractory advanced melanoma: a randomised dose-comparison cohort of a phase 1 trial. Lancet 384:1109–1117CrossRefPubMedGoogle Scholar
  23. 23.
    Kaufman HL, Kirkwood JM, Hodi FS et al (2013) The Society for Immunotherapy of Cancer Consensus statement on tumour immunotherapy for the treatment of cutaneous melanoma. Nat Rev Clin Oncol 10:588–598CrossRefPubMedGoogle Scholar
  24. 24.
    Eisenhauer EA, Therasse P, Bogaerts J et al (2009) New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1). Eur J Cancer 45:228–247CrossRefPubMedGoogle Scholar
  25. 25.
    Kurtz DM, Gambhir SS (2014) Tracking cellular and immune therapies in cancer. Adv Cancer Res 124:257–296CrossRefPubMedGoogle Scholar
  26. 26.
    Ahmadzadeh M, Johnson LA, Heemskerk B et al (2009) Tumor antigen-specific CD8 T cells infiltrating the tumor express high levels of PD-1 and are functionally impaired. Blood 114:1537–1544CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Chapon M, Randriamampita C, Maubec E et al (2011) Progressive upregulation of PD-1 in primary and metastatic melanomas associated with blunted TCR signaling in infiltrating T lymphocytes. J Invest Dermatol 131:1300–1307CrossRefPubMedGoogle Scholar
  28. 28.
    French JD, Kotnis GR, Said S et al (2012) Programmed death-1+ T cells and regulatory T cells are enriched in tumor-involved lymph nodes and associated with aggressive features in papillary thyroid cancer. J Clin Endocrinol Metab 97:E934–E943CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Lee CM, Tannock IF (2010) The distribution of the therapeutic monoclonal antibodies cetuximab and trastuzumab within solid tumors. BMC Cancer 10:255–266CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Matsuzaki J, Gnjatic S, Mhawech-Fauceglia P et al (2010) Tumor-infiltrating NY-ESO-1-specific CD8+ T cells are negatively regulated by LAG-3 and PD-1 in human ovarian cancer. Proc Natl Acad Sci U S A 107:7875–7880CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Muenst S, Soysal SD, Gao F et al (2013) The presence of programmed death 1 (PD-1)-positive tumor-infiltrating lymphocytes is associated with poor prognosis in human breast cancer. Breast Cancer Res Treat 139:667–676CrossRefPubMedGoogle Scholar
  32. 32.
    Scott AM, Wolchok JD, Old LJ (2012) Antibody therapy of cancer. Nat Rev Cancer 12:278–287CrossRefPubMedGoogle Scholar
  33. 33.
    Sfanos KS, Bruno TC, Meeker AK et al (2009) Human prostate-infiltrating CD8+ T lymphocytes are oligoclonal and PD-1+. Prostate 69:1694–1703CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Sun S, Fei X, Mao Y et al (2014) PD-1(+) immune cell infiltration inversely correlates with survival of operable breast cancer patients. Cancer Immunol Immunother 63:395–406CrossRefPubMedGoogle Scholar
  35. 35.
    Zhang Y, Huang S, Gong D et al (2010) Programmed death-1 upregulation is correlated with dysfunction of tumor-infiltrating CD8+ T lymphocytes in human non-small cell lung cancer. Cell Mol Immunol 7:389–395CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Shi F, Shi M, Zeng Z et al (2011) PD-1 and PD-L1 upregulation promotes CD8(+) T-cell apoptosis and postoperative recurrence in hepatocellular carcinoma patients. Int J Cancer 128:887–896CrossRefPubMedGoogle Scholar
  37. 37.
    Taube JM, Klein A, Brahmer JR et al (2014) Association of PD-1, PD-1 ligands, and other features of the tumor immune microenvironment with response to anti-PD-1 therapy. Clin Cancer Res 20:5064–5074CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    McDermott DF, Drake CG, Sznol M et al (2015) Survival, durable response, and long-term safety in patients with previously treated advanced renal cell carcinoma receiving nivolumab. J Clin Oncol 33:2013–2020CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Natarajan A, Habte F, Gambhir SS (2012) Development of a novel long-lived immunoPET tracer for monitoring lymphoma therapy in a humanized transgenic mouse model. Bioconjug Chem 23:1221–1229CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Lindmo T, Boven E, Cuttitta F et al (1984) Determination of the immunoreactive fraction of radiolabeled monoclonal antibodies by linear extrapolation to binding at infinite antigen excess. J Immunol Methods 72:77–89CrossRefPubMedGoogle Scholar
  41. 41.
    Natarajan A, Gambhir SS (2015) Radiation dosimetry study of [(89)Zr]rituximab tracer for clinical translation of B cell NHL imaging using positron emission tomography. Mol Imaging Biol 17:539–547CrossRefPubMedPubMedCentralGoogle Scholar
  42. 42.
    Suffner J, Hochweller K, Kuhnle MC et al (2010) Dendritic cells support homeostatic expansion of Foxp3+ regulatory T cells in Foxp3.LuciDTR mice. J Immunol 184:1810–1820CrossRefPubMedGoogle Scholar
  43. 43.
    Khoja L, Butler MO, Kang SP et al (2015) Pembrolizumab. J Immunother Cancer 3:36–49CrossRefPubMedPubMedCentralGoogle Scholar
  44. 44.
    Amadori A, Veronesi A, Coppola V et al (1996) The hu-PBL-SCID mouse in human lymphocyte function and lymphomagenesis studies: achievements and caveats. Semin Immunol 8:249–254CrossRefPubMedGoogle Scholar
  45. 45.
    Greiner DL, Hesselton RA, Shultz LD (1998) SCID mouse models of human stem cell engraftment. Stem Cells 16:166–177CrossRefPubMedGoogle Scholar
  46. 46.
    Legrand N, Weijer K, Spits H (2006) Experimental models to study development and function of the human immune system in vivo. J Immunol 176:2053–2058CrossRefPubMedGoogle Scholar
  47. 47.
    King M, Pearson T, Shultz LD et al (2008) A new Hu-PBL model for the study of human islet alloreactivity based on NOD-scid mice bearing a targeted mutation in the IL-2 receptor gamma chain gene. Clin Immunol 126:303–314CrossRefPubMedGoogle Scholar
  48. 48.
    Brehm MA, Shultz LD, Greiner DL (2010) Humanized mouse models to study human diseases. Curr Opin Endocrinol Diabetes Obes 17:120–125CrossRefPubMedPubMedCentralGoogle Scholar
  49. 49.
    Roth MD, Harui A (2015) Human tumor infiltrating lymphocytes cooperatively regulate prostate tumor growth in a humanized mouse model. J Immunother Cancer 3:12–33CrossRefPubMedPubMedCentralGoogle Scholar
  50. 50.
    Sharma P, Allison JP (2015) Immune checkpoint targeting in cancer therapy: toward combination strategies with curative potential. Cell 161:205–214CrossRefPubMedGoogle Scholar
  51. 51.
    Krishnan C, Warnke RA, Arber DA et al (2010) PD-1 expression in T-cell lymphomas and reactive lymphoid entities: potential overlap in staining patterns between lymphoma and viral lymphadenitis. Am J Surg Pathol 34:178–189CrossRefPubMedGoogle Scholar
  52. 52.
    England CG, Ehlerding EB, Hernandez R et al (2016) Preclinical pharmacokinetics and biodistribution studies of 89Zr-labeled pembrolizumab. J Nucl Med 58:162–168CrossRefPubMedGoogle Scholar
  53. 53.
    Natarajan A, Mayer AT, Xu L et al (2015) Novel radiotracer for ImmunoPET imaging of PD-1 checkpoint expression on tumor infiltrating lymphocytes. Bioconjug Chem 26:2062–2069CrossRefPubMedGoogle Scholar

Copyright information

© World Molecular Imaging Society 2017

Authors and Affiliations

  • Arutselvan Natarajan
    • 1
  • Aaron T. Mayer
    • 1
    • 2
  • Robert E. Reeves
    • 1
  • Claude M. Nagamine
    • 3
  • Sanjiv Sam Gambhir
    • 1
    • 2
    • 4
    • 5
  1. 1.Department of Radiology, School of MedicineStanford UniversityStanfordUSA
  2. 2.Department of BioengineeringStanford UniversityStanfordUSA
  3. 3.Department of Comparative MedicineStanford UniversityStanfordUSA
  4. 4.Department of Materials Science and EngineeringStanford UniversityStanfordUSA
  5. 5.Molecular Imaging Program at Stanford, Department of RadiologyStanford UniversityStanfordUSA

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