18F-labeled anti-human CD20 cys-diabody for same-day immunoPET in a model of aggressive B cell lymphoma in human CD20 transgenic mice

  • Kirstin A. ZettlitzEmail author
  • Richard Tavaré
  • Wen-Ting K. Tsai
  • Reiko E. Yamada
  • Noel S. Ha
  • Jeffrey Collins
  • R. Michael van Dam
  • John M. Timmerman
  • Anna M. Wu
Original Article



Metabolic imaging using [18F]FDG is the current standard for clinical PET; however, some malignancies (e.g., indolent lymphomas) show low avidity for FDG. The majority of B cell lymphomas express CD20, making it a valuable target both for antibody-based therapy and imaging. We previously developed PET tracers based on the humanised anti-CD20 antibody obinutuzumab (GA101). Preclinical studies showed that the smallest bivalent fragment, the cys-diabody (GAcDb, 54.5 kDa) with a peak uptake at 1–2 h post-injection and a biological half-life of 2–5 h, is compatible with short-lived positron emitters such as fluorine-18 (18F, t1/2 110 min), enabling same-day imaging.


GAcDb was radiolabeled using amine-reactive N-succinimidyl 4-[18F]-fluorobenzoate ([18F]SFB), or thiol-reactive N-[2-(4-[18F]-fluorobenzamido)ethyl]maleimide ([18F]FBEM) for site-specific conjugation to C-terminal cysteine residues. Both tracers were used for immunoPET imaging of the B cell compartment in human CD20 transgenic mice (hCD20TM). [18F]FB-GAcDb immunoPET was further evaluated in a disseminated lymphoma (A20-hCD20) syngeneic for hCD20TM and compared to [18F]FDG PET. Tracer uptake was confirmed by ex vivo biodistribution.


The GAcDb was successfully 18F-radiolabeled using two different conjugation methods resulting in similar specific activities and without impairing immunoreactivity. Both tracers ([18F]FB-GAcDb and [18F]FBEM-GAcDb) specifically target human CD20-expressing B cells in transgenic mice. Fast blood clearance results in high contrast PET images as early as 1 h post injection enabling same-day imaging. [18F]FB-GAcDb immunoPET detects disseminated lymphoma disease in the context of normal tissue expression of hCD20, with comparable sensitivity as [18F]FDG PET but with added specificity for the therapeutic target.


[18F]FB-GAcDb and [18F]FBEM-GAcDb could monitor normal B cells and B cell malignancies non-invasively and quantitatively in vivo. In contrast to [18F]FDG PET, immunoPET provides not only information about the extent of disease but also about presence and localisation of the therapeutic target.


Anti-CD20 cys-diabody Antibody fragments ImmunoPET 18B cell lymphoma Same-day imaging 



The authors thank Felix B Salazar for technical support and the Preclinical Imaging Center (UCLA) for help with small-animal PET scans.


This work was supported by NIH grant CA149254, NIH grant CA212718 and by a generous gift from Ralph and Marjorie Crump to the Crump Institute for Molecular Imaging. Small-animal imaging and flow cytometry were funded in part by the UCLA Jonsson Comprehensive Cancer Center (JCCC) Support Grant (NIH CA016042). AM Wu, JM Timmerman and RM van Dam are members of the JCCC.

Compliance with ethical standards

Conflict of interest

JM Timmerman reports receiving commercial research grants from Bristol-Myers Squibb, Kite Pharma, and Valor Biotherapeutics, and is a consultant/ advisory board member for Celgene and Seattle Genetics. AM Wu holds ownership interest in and is a consultant/advisory board member for ImaginAb, Inc. No potential conflicts of interest were disclosed by the other authors.

Ethical approval

All procedures performed in studies involving animals were in accordance with the ethical standards of the University of California Los Angeles (UCLA) Animal Research Committee. This article does not contain any studies with human participants performed by any of the authors.

Supplementary material

259_2018_4214_MOESM1_ESM.docx (236 kb)
ESM 1 (DOCX 236 kb)


  1. 1.
    Barrington SF, Johnson PWM. 18F-FDG PET/CT in lymphoma: has imaging-directed personalized medicine become a reality? J Nucl Med. 2017;58(10):1539–44.PubMedPubMedCentralGoogle Scholar
  2. 2.
    Barrington SF, Mikhaeel NG, Kostakoglu L, Meignan M, Hutchings M, Mueller SP, et al. Role of imaging in the staging and response assessment of lymphoma: consensus of the International Conference on Malignant Lymphomas Imaging Working Group. J Clin Oncol. 2014;32(27):3048–58.PubMedPubMedCentralGoogle Scholar
  3. 3.
    Meignan M, Itti E, Gallamini A, Younes A. FDG PET/CT imaging as a biomarker in lymphoma. Eur J Nucl Med Mol Imaging. 2015;42(4):623–33.PubMedGoogle Scholar
  4. 4.
    Adams HJ, Nievelstein RA, Kwee TC. Systematic Review on the Additional Value of 18F-Fluoro-2-Deoxy-D-Glucose Positron Emission Tomography in Staging Follicular Lymphoma. J Comput Assist Tomogr. 2017;41(1):98–103.PubMedGoogle Scholar
  5. 5.
    Kostakoglu L, Cheson BD. Current role of FDG PET/CT in lymphoma. Eur J Nucl Med Mol Imaging. 2014;41(5):1004–27.PubMedGoogle Scholar
  6. 6.
    Sun N, Zhao J, Qiao W, Wang T. Predictive value of interim PET/CT in DLBCL treated with R-CHOP: meta-analysis. Biomed Res Int. 2015;2015:648572.PubMedPubMedCentralGoogle Scholar
  7. 7.
    England CG, Rui L, Cai W. Lymphoma: current status of clinical and preclinical imaging with radiolabeled antibodies. Eur J Nucl Med Mol Imaging. 2017;44(3):517–32.PubMedGoogle Scholar
  8. 8.
    Tang J, Salloum D, Carney B, Brand C, Kossatz S, Sadique A, et al. Targeted PET imaging strategy to differentiate malignant from inflamed lymph nodes in diffuse large B-cell lymphoma. Proc Natl Acad Sci USA. 2017;114(36):E7441–E9.PubMedGoogle Scholar
  9. 9.
    Friedberg JW, Chengazi V. PET scans in the staging of lymphoma: current status. Oncologist. 2003;8(5):438–47.PubMedGoogle Scholar
  10. 10.
    Goldsmith SJ. Radioimmunotherapy of lymphoma: Bexxar and Zevalin. Semin Nucl Med. 2010;40(2):122–35.PubMedGoogle Scholar
  11. 11.
    Lim SH, Beers SA, French RR, Johnson PW, Glennie MJ, Cragg MS. Anti-CD20 monoclonal antibodies: historical and future perspectives. Haematologica. 2010;95(1):135–43.PubMedGoogle Scholar
  12. 12.
    Du FH, Mills EA, Mao-Draayer Y. Next-generation anti-CD20 monoclonal antibodies in autoimmune disease treatment. Auto Immun Highlights. 2017;8(1):12.PubMedPubMedCentralGoogle Scholar
  13. 13.
    Natarajan A, Habte F, Gambhir SS. Development of a novel long-lived immunoPET tracer for monitoring lymphoma therapy in a humanized transgenic mouse model. Bioconjug Chem. 2012;23(6):1221–9.PubMedPubMedCentralGoogle Scholar
  14. 14.
    Muylle K, Flamen P, Vugts DJ, Guiot T, Ghanem G, Meuleman N, et al. Tumour targeting and radiation dose of radioimmunotherapy with (90)Y-rituximab in CD20+ B-cell lymphoma as predicted by (89)Zr-rituximab immuno-PET: impact of preloading with unlabelled rituximab. Eur J Nucl Med Mol Imaging. 2015;42(8):1304–14.PubMedPubMedCentralGoogle Scholar
  15. 15.
    Zettlitz KA, Tavare R, Knowles SM, Steward KK, Timmerman JM, Wu AM. ImmunoPET of malignant and normal B cells with 89Zr- and 124I-labeled obinutuzumab antibody fragments reveals differential CD20 internalization in vivo. Clin Cancer Res. 2017;23(23):7242–7252.Google Scholar
  16. 16.
    Williams LE, Wu AM, Yazaki PJ, Liu A, Raubitschek AA, Shively JE, et al. Numerical selection of optimal tumor imaging agents with application to engineered antibodies. Cancer Biother Radiopharm. 2001;16(1):25–35.PubMedGoogle Scholar
  17. 17.
    Kiesewetter DO, Jacobson O, Lang L, Chen X. Automated radiochemical synthesis of [18F]FBEM: a thiol reactive synthon for radiofluorination of peptides and proteins. Appl Radiat Isot. 2011;69(2):410–4.PubMedGoogle Scholar
  18. 18.
    Collins J, Waldmann CM, Drake C, Slavik R, Ha NS, Sergeev M, et al. Production of diverse PET probes with limited resources: 24 (18)F-labeled compounds prepared with a single radiosynthesizer. Proc Natl Acad Sci USA. 2017;114(43):11309–14.PubMedGoogle Scholar
  19. 19.
    Vaidyanathan G, Zalutsky MR. Labeling proteins with fluorine-18 using N-succinimidyl 4-[18F]fluorobenzoate. Int J Rad Appl Instrum B. 1992;19(3):275–81.PubMedGoogle Scholar
  20. 20.
    Cai W, Olafsen T, Zhang X, Cao Q, Gambhir SS, Williams LE, et al. PET imaging of colorectal cancer in xenograft-bearing mice by use of an 18F-labeled T84.66 anti-carcinoembryonic antigen diabody. J Nucl Med. 2007;48(2):304–10.PubMedGoogle Scholar
  21. 21.
    Liu K, Lepin EJ, Wang MW, Guo F, Lin WY, Chen YC, et al. Microfluidic-based 18F-labeling of biomolecules for immuno-positron emission tomography. Mol Imaging. 2011;10(3):168–76 1-7.PubMedPubMedCentralGoogle Scholar
  22. 22.
    Sharma SK, Wuest M, Way JD, Bouvet VR, Wang M, Wuest FR. Synthesis and pre-clinical evaluation of an (18)F-labeled single-chain antibody fragment for PET imaging of epithelial ovarian cancer. Am J Nucl Med Mol Imaging. 2016;6(3):185–98.PubMedPubMedCentralGoogle Scholar
  23. 23.
    Vaidyanathan G, Zalutsky MR. Synthesis of N-succinimidyl 4-[18F]fluorobenzoate, an agent for labeling proteins and peptides with 18F. Nat Protoc. 2006;1(4):1655–61.PubMedGoogle Scholar
  24. 24.
    Vaidyanathan G, McDougald D, Choi J, Koumarianou E, Weitzel D, Osada T, et al. Preclinical Evaluation of 18F-Labeled Anti-HER2 Nanobody Conjugates for Imaging HER2 Receptor Expression by Immuno-PET. J Nucl Med. 2016;57(6):967–73.PubMedPubMedCentralGoogle Scholar
  25. 25.
    Cai W, Zhang X, Wu Y, Chen X. A thiol-reactive 18F-labeling agent, N-[2-(4-18F-fluorobenzamido)ethyl]maleimide, and synthesis of RGD peptide-based tracer for PET imaging of alpha v beta 3 integrin expression. J Nucl Med. 2006;47(7):1172–80.PubMedPubMedCentralGoogle Scholar
  26. 26.
    Kramer-Marek G, Kiesewetter DO, Martiniova L, Jagoda E, Lee SB, Capala J. [18F]FBEM-Z(HER2:342)-Affibody molecule-a new molecular tracer for in vivo monitoring of HER2 expression by positron emission tomography. Eur J Nucl Med Mol Imaging. 2008;35(5):1008–18.PubMedGoogle Scholar
  27. 27.
    Li W, Niu G, Lang L, Guo N, Ma Y, Kiesewetter DO, et al. PET imaging of EGF receptors using [18F]FBEM-EGF in a head and neck squamous cell carcinoma model. Eur J Nucl Med Mol Imaging. 2012;39(2):300–8.PubMedGoogle Scholar
  28. 28.
    Johnson NA, Savage KJ, Ludkovski O, Ben-Neriah S, Woods R, Steidl C, et al. Lymphomas with concurrent BCL2 and MYC translocations: the critical factors associated with survival. Blood. 2009;114(11):2273–9.PubMedPubMedCentralGoogle Scholar
  29. 29.
    Li S, Lin P, Young KH, Kanagal-Shamanna R, Yin CC, Medeiros LJ. MYC/BCL2 double-hit high-grade B-cell lymphoma. Adv Anat Pathol. 2013;20(5):315–26.PubMedGoogle Scholar
  30. 30.
    Ahuja A, Shupe J, Dunn R, Kashgarian M, Kehry MR, Shlomchik MJ. Depletion of B cells in murine lupus: efficacy and resistance. J Immunol. 2007;179(5):3351–61.PubMedGoogle Scholar
  31. 31.
    Kim KJ, Kanellopoulos-Langevin C, Merwin RM, Sachs DH, Asofsky R. Establishment and characterization of BALB/c lymphoma lines with B cell properties. J Immunol. 1979;122(2):549–54.PubMedGoogle Scholar
  32. 32.
    Chaise C, Itti E, Petegnief Y, Wirquin E, Copie-Bergman C, Farcet JP, et al. [F-18]-Fluoro-2-deoxy-D: -glucose positron emission tomography as a tool for early detection of immunotherapy response in a murine B cell lymphoma model. Cancer Immunol Immunother. 2007;56(8):1163–71.PubMedGoogle Scholar
  33. 33.
    Lazari M, Lyashchenko SK, Burnazi EM, Lewis JS, van Dam RM, Murphy JM. Fully-automated synthesis of 16beta-(18)F-fluoro-5alpha-dihydrotestosterone (FDHT) on the ELIXYS radiosynthesizer. Appl Radiat Isot. 2015;103:9–14.PubMedPubMedCentralGoogle Scholar
  34. 34.
    Bhatt S, Parvin S, Zhang Y, Cho HM, Kunkalla K, Vega F, et al. Anti-CD20-interleukin-21 fusokine targets malignant B cells via direct apoptosis and NK-cell-dependent cytotoxicity. Blood. 2017;129(16):2246–56.PubMedGoogle Scholar
  35. 35.
    Loening AM, Gambhir SS. AMIDE: a free software tool for multimodality medical image analysis. Mol Imaging. 2003;2(3):131–7.PubMedGoogle Scholar
  36. 36.
    Waldmann CM, Gomez A, Marchis P, Bailey ST, Momcilovic M, Jones AE, et al. An Automated Multidose Synthesis of the Potentiometric PET Probe 4-[(18)F]Fluorobenzyl-Triphenylphosphonium ([(18)F]FBnTP). Mol Imaging Biol. 2018;20(2):205–12.PubMedPubMedCentralGoogle Scholar
  37. 37.
    Christensen EI, Nielsen S. Structural and functional features of protein handling in the kidney proximal tubule. Semin Nephrol. 1991;11(4):414–39.PubMedGoogle Scholar
  38. 38.
    Nielsen S. Endocytosis in proximal tubule cells involves a two-phase membrane-recycling pathway. Am J Phys. 1993;264(4 Pt 1):C823–35.Google Scholar
  39. 39.
    Vegt E, Wetzels JF, Russel FG, Masereeuw R, Boerman OC, van Eerd JE, et al. Renal uptake of radiolabeled octreotide in human subjects is efficiently inhibited by succinylated gelatin. J Nucl Med. 2006;47(3):432–6.PubMedGoogle Scholar
  40. 40.
    van Eerd JE, Vegt E, Wetzels JF, Russel FG, Masereeuw R, Corstens FH, et al. Gelatin-based plasma expander effectively reduces renal uptake of 111In-octreotide in mice and rats. J Nucl Med. 2006;47(3):528–33.PubMedGoogle Scholar
  41. 41.
    Akizawa H, Uehara T, Arano Y. Renal uptake and metabolism of radiopharmaceuticals derived from peptides and proteins. Adv Drug Deliv Rev. 2008;60(12):1319–28.PubMedGoogle Scholar
  42. 42.
    MartIn-Fontecha A, Sebastiani S, Hopken UE, Uguccioni M, Lipp M, Lanzavecchia A, et al. Regulation of dendritic cell migration to the draining lymph node: impact on T lymphocyte traffic and priming. J Exp Med. 2003;198(4):615–21.PubMedPubMedCentralGoogle Scholar
  43. 43.
    Ginaldi L, De Martinis M, Matutes E, Farahat N, Morilla R, Catovsky D. Levels of expression of CD19 and CD20 in chronic B cell leukaemias. J Clin Pathol. 1998;51(5):364–9.PubMedPubMedCentralGoogle Scholar
  44. 44.
    Briones J, Timmerman J, Levy R. In vivo antitumor effect of CD40L-transduced tumor cells as a vaccine for B-cell lymphoma. Cancer Res. 2002;62(11):3195–9.PubMedGoogle Scholar
  45. 45.
    Curti A, Parenza M, Colombo MP. Autologous and MHC class I-negative allogeneic tumor cells secreting IL-12 together cure disseminated A20 lymphoma. Blood. 2003;101(2):568–75.PubMedGoogle Scholar
  46. 46.
    Siegel S, Wagner A, Schmitz N, Zeis M. Induction of antitumour immunity using survivin peptide-pulsed dendritic cells in a murine lymphoma model. Br J Haematol. 2003;122(6):911–4.PubMedGoogle Scholar
  47. 47.
    Passineau MJ, Siegal GP, Everts M, Pereboev A, Jhala D, Wang M, et al. The natural history of a novel, systemic, disseminated model of syngeneic mouse B-cell lymphoma. Leuk Lymphoma. 2005;46(11):1627–38.PubMedGoogle Scholar
  48. 48.
    Pals ST, de Gorter DJ, Spaargaren M. Lymphoma dissemination: the other face of lymphocyte homing. Blood. 2007;110(9):3102–11.PubMedGoogle Scholar
  49. 49.
    Kennedy GA, Tey SK, Cobcroft R, Marlton P, Cull G, Grimmett K, et al. Incidence and nature of CD20-negative relapses following rituximab therapy in aggressive B-cell non-Hodgkin’s lymphoma: a retrospective review. Br J Haematol. 2002;119(2):412–6.PubMedGoogle Scholar
  50. 50.
    Hiraga J, Tomita A, Sugimoto T, Shimada K, Ito M, Nakamura S, et al. Down-regulation of CD20 expression in B-cell lymphoma cells after treatment with rituximab-containing combination chemotherapies: its prevalence and clinical significance. Blood. 2009;113(20):4885–93.PubMedGoogle Scholar
  51. 51.
    Kennedy AD, Beum PV, Solga MD, DiLillo DJ, Lindorfer MA, Hess CE, et al. Rituximab Infusion Promotes Rapid Complement Depletion and Acute CD20 Loss in Chronic Lymphocytic Leukemia. J Immunol. 2004;172(5):3280–8.PubMedGoogle Scholar
  52. 52.
    Beers SA, French RR, Chan HT, Lim SH, Jarrett TC, Vidal RM, et al. Antigenic modulation limits the efficacy of anti-CD20 antibodies: implications for antibody selection. Blood. 2010;115(25):5191–201.PubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Kirstin A. Zettlitz
    • 1
    • 2
    Email author
  • Richard Tavaré
    • 1
    • 3
  • Wen-Ting K. Tsai
    • 1
  • Reiko E. Yamada
    • 4
  • Noel S. Ha
    • 1
    • 5
  • Jeffrey Collins
    • 1
  • R. Michael van Dam
    • 1
    • 5
  • John M. Timmerman
    • 4
  • Anna M. Wu
    • 1
    • 2
  1. 1.Crump Institute for Molecular Imaging, Department of Molecular and Medical Pharmacology, David Geffen School of MedicineUniversity of California, Los AngelesLos AngelesUSA
  2. 2.Department of Molecular Imaging and TherapyBeckman Research Institute, City of HopeDuarteUSA
  3. 3.Regeneron Pharmaceuticals, IncTarrytownUSA
  4. 4.Division of Hematology & Oncology, Department of MedicineUniversity of California, Los AngelesLos AngelesUSA
  5. 5.Department of Bioengineering, Samueli School of EngineeringUniversity of California, Los AngelesLos AngelesUSA

Personalised recommendations