In Situ Vaccination with Combination of Class B and Class C Toll-like Receptor 9 Agonist CpG Immune Adjuvant Nanoparticles to Induce a Systemic Anti-Lymphoma Response
Authors: Lin Adam Yuh , Rink Jonathan Scott , Thaxton Colby Shad , Gordon Leo I. 
Published: November 2021

Abstract
Introduction: Treatment of relapsed and refractory aggressive B cell lymphomas is challenging, especially after autologous stem cell transplant or chimeric antigen receptor T cell therapy. CpG oligodeoxynucleotides (CpGs) mimic bacterial DNA and bind to toll-like receptor 9 (TLR9). TLR9 is expressed in the endosomes of innate immune cells and B lymphocytes as well as B cell lymphomas. CpG binding to TLR9 in these cells leads to a pro-inflammatory response and induces apoptosis of lymphoma cells by altering NF-kB activation. However, clinical utility of CpGs is limited due to difficulties delivering the DNA directly to the innate immune cells and lymphoma cells, and these DNA strands are easily degraded in biological fluids. Therefore, we designed a nano-carrier to deliver two classes of CpGs to improve clinical efficacy of CpGs in treating lymphoma. Class B CpGs (B-CpGs) mainly stimulate B cells while class C CpGs (C-CpGs) act on both B cells and plasmacytoid dendritic cells. Though C-CpGs are the focus of current clinical trials, B-CpGs may have a special a role in the treatment of lymphoma due to their more potent, direct cytotoxic effect on the malignant lymphoma cells. Here, we evaluated the anti-lymphoma effect of the combination of B-CpG nanoparticles (BNP) and C-CpG nanoparticles (CNP) by optimizing lymphoma cell death and immune stimulation.
Methods: BNP and CNP were synthesized by surface-functionalizing gold nanoparticles with modified B-CpGs and C-CpGs, followed by purification via centrifugation. We used a dual flank tumor murine lymphoma model to evaluate the local and systemic anti-lymphoma efficacy of the CpG NPs and screen for the most effective formulation. A20 lymphoma tumors were implanted on both flanks of Balb/c mice. When the tumors were 5-7mm in diameter, various treatments were injected intratumorally on days 1, 4, and 8 on the side with the larger tumor. Treatment groups include PBS, B-CpG, C-CpG, B+C-CpG, BNP, CNP, BNP+CNP. Free CpGs were injected at 50ug/ml in 50ul (2.5ug CpG per injection) and nanoparticles at 100nM (equivalent to 50ug/ml CpGs) in 50ul. Tumor growth were measured every other day. Event is defined as tumor volume &gt; 3cm 3. Second, we used an advanced disease lymphoma model to evaluate intravenous delivery of combination NPs. We implanted A20 lymphoma cells on a flank tumor (on day -14), then we injected A20 cells intravenously to mimic advanced disease (on day -7). We treated the mice with intravenous (tail vein) injections of PBS, B+C-CpG, or BNP+CNP on days 1, 4, and 8.
Results: We found that the combination of BNP and CNPs had the most significant reduction in total tumor volume (TTV) (treated + untreated tumor size) (393mm 3 on day 22). The PBS group demonstrated rapid tumor growth (TTV 3516mm 3). The experimental treatment groups, B-CpG (TTV 949mm 3),, C-CpG (TTV 2230mm 3), B+C-CpGs (TTV 1680mm 3), BNP (TTV 1224mm 3), and CNP (1894mm 3) had similar TTV growth curves. Separating the treated tumor and untreated tumor growth curves, we found that B-CpG and BNP along with combination of BNP+CNP had the best tumor suppression on the treated side. This is not surprising as B-CpG sequences are more directly cytotoxic to lymphoma cells versus C-CpGs. Interestingly, combination nanoparticle treatment (BNP+CNP) was the only group that had significantly reduced tumor growth of the untreated tumor compared with all other conditions (p&lt;0.03) where no statistical difference was measured when the PBS treatment group with the other groups. The BNP+CNP treatment group also had significantly prolonged event free survival compared with all other treatment groups. Furthermore, in the advanced disease model, when BNP and CNP were both injected intravenously, the flank tumor was notably smaller (149mm 3) compared with the other two groups (B+C-CpG 1310mm 3 or PBS 1555mm 3). Importantly, there were no visible lymphoma nodules on liver in the nanoparticle treatment group while there are obvious nodules for both the PBS and the free CpG groups.
Conclusions: Overall, the combination of class B and class C CpG nanoparticles can generate a strong in situ anti-lymphoma vaccination response, presumably through the combination of by combining the superior cytotoxicity of BNPs with the enhanced immune stimulation effect of the CNPs. Further evaluation of the combined mechanism of action of this dual CpG nanoparticle approach and toxicity studies are in progress.

Disclosures
Thaxton: Zylem biosciences: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Gordon: Zylem Biosciences: Patents & Royalties: Patents, No royalties; Bristol Myers Squibb: Honoraria, Research Funding.