Intratumoral Injection of CpG-ODN Plus Systemic Ibrutinib Induces an Anti-Tumor Immune Response Affecting T Cell Subsets in the Microenvironment of Both Injected and Non-Injected Tumor Sites in Patients with Low-Grade Lymphoma
Authors: Czerwinski Debra K. , Frank Matthew J. , Shree Tanaya , Khodadoust Michael S , Long Steven R. , Levy Ronald 
Published: October 2019

Abstract
BACKGROUND: Low-grade B cell lymphoma is often characterized by an infiltration of immune effector cells including T, NK and dendritic cells. But, despite the presence of effector cells within the tumor, these cells fail to control tumor growth.
In a preclinical mouse model, we showed that Ibrutinib, an inhibitor of Bruton's tyrosine kinase (BTK), but also ITK (IL-2-inducible T cell kinase), synergized with intratumoral CpG to facilitate complete regression of tumors at the treated site as well as a distal, non-treated site, curing all the mice. This was accompanied by a potent anti-tumor memory T cell response by both CD4 and CD8 T cells that rejected the tumor on re-challenge. (Sagiv-Barfi I, et al. Blood. 2015 March:125(13):2079-2086)
STUDY: In an ongoing clinical trial (NCT02927964), patients with previously treated low-grade lymphoma receive low dose (2Gyx2) radiotherapy to a single tumor site followed by 5 weekly intratumoral injections of 3 mg CpG-ODN (SD-101, Dynavax Technologies) into the same site. 1 day after the second injection, patients begin taking a daily 560 mg dose of Ibrutinib. A fine needle aspirate (FNA) of the injected site and a non-injected site outside the radiation field is performed prior to radiotherapy, one week after the first injection, and at week 6, 1 week after the final injection of CpG. FNA samples are stained for flow cytometry with panels of antibodies to delineate all major cell populations and their subsets. Cellular activation as well as T cell exhaustion, inhibition and function are also characterized. When feasible, a biopsy is performed prior to treatment providing tumor cells to be used in an immune response assay to evaluate induced anti-tumor responses by circulating peripheral blood T cells obtained throughout the study.
RESULTS: To date, 12 patients have been entered onto the study. Of these patients, 4 had excisional biopsies and subsequent immune response assays performed. All 4 patients exhibited CD8 anti-tumor responses as determined by an increase in the activation marker CD137 as well as the functional marker granzyme B above pretreatment CD8 T cells. At week 12, 7 weeks after the last CpG injection, the average increase above pretreatment baseline for CD137 and granzyme B was 6.1% and 9.3%, respectively. In 3 of these patients, we observed an increase in CD8 T cells expressing CD137 and granzyme B from the FNA of the non-injected site. The 4th patient did not have adequate number of cells for staining. Two patients exhibited CD4 immune responses as characterized by an up-regulation of CD137 and CD278.
FNA of 7 patients produced enough cells to analyze the tumor microenvironment from both the injected and the non-injected sites over all 3 time points; 1 patient was evaluable through week 2 for both sites. In 7 out of these 8 patients, CD3 T cells increased at the injected and non-injected sites by week 6. The proportion of CD4 and CD8 T cells did not stay constant, however, as reflected by the changes in CD4:CD8 ratios. This suggests that the increase in T cells was not purely the result of a loss of tumor B cells in the samples. Notably, we observed a significant increase in the effector CD4 T cells in all patients at the injected site by week 2 (14.1% ± 6.5%, p = 0.005) and 5 of 8 patients in the non-injected site by week 6 (12.4% ± 10.4%). Moreover, the proportion of T follicular helper cells significantly decreased in all patients at the treated site by week 2 (17.7% ± 9.7% of T cells, p = 0.0012) and in 5 of 8 patients at the non-treated site by week 6 (8.1% ± 5.5% of T cells). Tregs were more variable, decreasing in 5 of 8 patients in the treated and 3 of 8 patients in the non-treated site by week 6.
CONCLUSION: CpG is known to activate antigen-presenting cells such as dendritic cells and macrophages. Ibrutinib is a small molecule that has been shown to have direct anti-tumor effects in B cell lymphoma and may skew an immune response towards that of a TH1 type. Here we show that together, they can effect changes in the tumor microenvironment in both treated and in untreated sites of disease.
This clinical trial is ongoing and open to accrual.

Disclosures
Khodadoust: Innate Pharma: Research Funding.