In situ vaccination with a TLR9 agonist induces systemic lymphoma regression: a phase I/II study

Abstract

Purpose: Combining tumor antigens with an immunostimulant can induce the immune system to specifically eliminate cancer cells. Generally, this combination is accomplished in an ex vivo, customized manner. In a preclinical lymphoma model, intratumoral injection of a Toll-like receptor 9 (TLR9) agonist induced systemic antitumor immunity and cured large, disseminated tumors.

Patients and methods: We treated 15 patients with low-grade B-cell lymphoma using low-dose radiotherapy to a single tumor site and-at that same site-injected the C-G enriched, synthetic oligodeoxynucleotide (also referred to as CpG) TLR9 agonist PF-3512676. Clinical responses were assessed at distant, untreated tumor sites. Immune responses were evaluated by measuring T-cell activation after in vitro restimulation with autologous tumor cells.

Results: This in situ vaccination maneuver was well-tolerated with only grade 1 to 2 local or systemic reactions and no treatment-limiting adverse events. One patient had a complete clinical response, three others had partial responses, and two patients had stable but continually regressing disease for periods significantly longer than that achieved with prior therapies. Vaccination induced tumor-reactive memory CD8 T cells. Some patients' tumors were able to induce a suppressive, regulatory phenotype in autologous T cells in vitro; these patients tended to have a shorter time to disease progression. One clinically responding patient received a second course of vaccination after relapse resulting in a second, more rapid clinical response.

Conclusion: In situ tumor vaccination with a TLR9 agonist induces systemic antilymphoma clinical responses. This maneuver is clinically feasible and does not require the production of a customized vaccine product.

Fig 1.

Intratumoral vaccination induces objective clinical responses. (A) Patients received 2 Gy × 2 radiation combined with intratumoral injection of PF-3512676 to a single disease site and disease was measured at up to six distant sites. (B) Waterfall plot showing percent change in the cross-product sum at the time of best response (indicated above ordinate) versus pretreatment. (*) Refers to patient 10, whose primary cutaneous disease was instead measured as a three-dimensional sum. SPD, sum of products of greatest diameter.

Fig 2.

Intratumoral vaccination induces objective clinical responses [extended]. (A) Complete response in patient 3, treated site: occipital; visualized site: bilateral axillae. (B) Partial response in patient 10, treated site: suprasternal cutaneous; visualized site: supra-orbital cutaneous.

Fig 3.

Intratumoral vaccination induces objective clinical responses [extended]. (A) Partial response (PR) in patient 11, treated site: right supraclavicular; visualized site: left submandibular. (B) PR in patient 12, treated site: left inguinal; visualized site: retroperitoneal lymph node conglomerate.

Fig 4.

Re-treatment of a responding patient. Patient 12 received re-treatment as in Figure 1 with intratumoral injection of PF-3512676 18 mg. (A) Partial response in patient 12, treated site: right inguinal; visualized sites: right supraclavicular and retroperitoneal lymph node conglomerate. (B) Increased proportion of tumor-reactive memory CD8 T cells per CD137 upregulation in pleural fluid after re-treatment; data shown are gated on live, CD8+ cells, statistics shown are percentage of CD45RO+CD137+ cells among that gate.

Fig 5.

Regulatory T-cell (Treg) induction by CpG-activated tumors. (A) Prevaccination peripheral blood lymphocytes were cultured with media or with autologous, CpG-activated tumor B-cells for 120 hours, then assessed per flow cytometry for CD4, CD25, and forkhead box protein P3 (FOXP3). Data shown are gated on live CD4+ cells; Treg fold-induction statistics shown are CD25+FOXP3+tumor/CD25+FOXP3+media. Progression-free survival (PFS) for each patient was correlated to Treg fold-induction using the latter as a (B) dichotomous variable (≤ 3-fold v > 3-fold) and comparing PFS by log-rank test, or (C) as a continuous variable and relating to PFS by Cox proportional hazard model.

Fig A1.

Immune responses. (A, B) CpG induction of immunogenic phenotype in patient tumor cells. Histograms represent coculture with PF-3512676 (green line) or media alone (red line). (C) Induction of tumor-reactive memory CD8 T cells per upregulation of CD137; data are gated on live CD8+ cells, statistics are percentages of CD45RO+CD137+ cells among that gate. (D) Induction of tumor-reactive CD8 T cells per interferon-γ (IFNg), interleukin-2 (IL2), and tumor necrosis factor (TNF) upregulation; data are gated on live CD8+ cells, statistics are percentages of cytokine(+) cells among that gate.