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 tumorand 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 CD137activated 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 antiCD20 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 antiCD137 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-g 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. Stanford University, Stanford, CA, USA Full list of author information is available at the end of the article Mueller et al. Journal for ImmunoTherapy of Cancer 2014, 2(Suppl 3):P106 http://www.immunotherapyofcancer.org/content/2/S3/P106