Skip to main content

Volume 2 Supplement 3

Abstracts of the 29th Annual Scientific Meeting of the Society for Immunotherapy of Cancer (SITC)

  • Poster presentation
  • Open access
  • Published:

Sequential tumor and dual immune targeted immunotherapy: anti-lymphoma activity of Rituximab with 4-1bb stimulation and PD-1 blockade

To select maximally-efficacious, minimally-toxic regimens of combination tumor- and immune-targeted therapy, preclinical testing in immune-competent models is required. We previously demonstrated 4-1bb(CD137) stimulation augmented the innate immune response mediated by CD137+activated natural killers (NK) cells and the subsequent CD8+ T cell adaptive immune response when administered after tumor-targeting, ADCC-competent, mAbs targeting CD20, HER2, and EGFR. As four ongoing clinical trials (NCT01471210, NCT01775631, NCT02110082, NCT01307267) investigate this strategy, the CD8+ T cell response stimulated by 4-1bb was determined pre-clinically to be further augmented by PD-1/PD-L1 blockade, and a clinical trial is planned (NCT02179918). Taken together, we hypothesized, sequential tumor-targeting with anti-CD20 mAb, Rituximab, followed by dual immune-targeting with anti-CD137 agonism and PD-1/PD-L1 blockade would be efficacious and tolerable due to non-overlapping immune mechanisms and toxicity profiles.

Preclinical modeling was performed in a therapeutic, syngenic, A20 lymphoma BALB/c model combining anti-CD20 mAb (IgG2a-18B12, delivered intraperitoneally, i.p. on d5) with agonistic anti-CD137 mAb (IgG2a-2A, i.p. d6) and anti-PD-1 mAb (IgG2a-RMPI-14, i.p. d6) with intratumoral (i.t.) and circulating (c.) immune responses phenotyped by flow cytometry and time of flight mass cytometry (CyTOF). Combination immunotherapy with anti-CD137 and anti-PD1 was superior to either monotherapy without anti-CD20 treatment in a dose-dependent manner (p<.001 tumor-growth, p < .001 survival), though no mice were cured long-term. When administered following anti-CD20 treatment, combination immunotherapy with anti-CD137 and anti-PD1 was superior to either monotherapy in a sequence-dependent manner (p<.001 tumor-growth, and p < .001 survival) with all mice cured long-term and protected from re-challenge when anti-CD20 preceded combination immunotherapy. Target expression was dynamic, as exemplified by highest CD137 expression on i.t.Tregs, followed by i.t.CD8+ T cells. Following anti-CD20 mAb treatment, 1) CD137 expression increased 5-10x on i.t.NKs and 2-6x increase on c.NKs; 2) PD-L1 expression increased on A20 tumor and i.t.CD8+ T cells, and minimally on i.t.NKs; and 3) PD-1 expression increased on i.t.Tregs, i.t.CD8+ T cells, and i.t.NKs. Treatment with anti-CD137 agonist and PD-1 blockade 1) increased the ratio of i.t.NKs/Tregs and c.CD8+ T cells/Tregs, and 2) increased i.t. and c.CD8+ T cell tumor-specific IFN-γ secretion and i.t. and c.NK cell degranulation. Laboratory and necropsy studies identified B cell lymphopenia and mild transaminitis which was notably marked with combination immunotherapy.

We conclude that sequential tumor-targeting followed by dual immune-targeting is highly-efficacious with predictable toxicity and should be considered for clinical translation to augment three therapies with only marginal activity as monotherapy in advanced, relapsed/refractory lymphoma.

Author information

Authors and Affiliations


Rights and permissions

Open Access  This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made.

The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

To view a copy of this licence, visit

The Creative Commons Public Domain Dedication waiver ( applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Mueller, A.M., Hebb, J., Sagiv-Barfi, I. et al. Sequential tumor and dual immune targeted immunotherapy: anti-lymphoma activity of Rituximab with 4-1bb stimulation and PD-1 blockade. j. immunotherapy cancer 2 (Suppl 3), P106 (2014).

Download citation

  • Published:

  • DOI: