Distinct immunological activation profiles of dSLIM® and ProMune® depend on their different structural context

Abstract

IntroductionDNA‐based TLR9 agonists are potent activators of the immune system. ProMune® and dSLIM® belong to different families of TLR9 agonists and both have been established as cancer immunotherapeutics in clinical proof‐of‐concept studies. Unfortunately, ProMune® failed in pivotal oncological trials. dSLIM®, the active ingredient of Lefitolimod (MGN1703), successfully finished a double‐blinded, placebo‐controlled phase II study in patients with advanced colorectal cancer, exhibiting improved progression‐free survival and durable disease control.MethodsTo explain the different systemic efficacies of dSLIM® and ProMune®, both TLR9 agonists and chimeric molecules thereof are analyzed side‐by‐side in a panel of in vitro assays for immune activation.Results and conclusionsIndeed, dSLIM® exposure results in an IFN‐α dependent broad activation of immune cells whereas ProMune® strongly stimulates B cells. Moreover, all functional effects of dSLIM® strictly depend on the presence of CG‐motifs within its dumbbell‐shaped, covalently closed structural context. Conversely, several immunological effects of ProMune® like IL‐8 secretion are independent of CG‐motifs and could be ascribed to the phosphorothioate‐modifications of its DNA backbone, which may have caused the side effects of ProMune® in clinical trials. Finally, we showed that the implementation of ProMune® (ODN2006) base sequence into the characteristic dSLIM® dumbbell form resulted in dSLIM2006 with all beneficial effects for immunostimulation combined from both TLR9 classes without any CG‐independent effects.

Collapse of the Plasmacytoid Dendritic Cell Compartment in Advanced Cutaneous Melanomas by Components of the Tumor Cell Secretome

“…PDC activation by TLR agonists may have clinical benefit in cancer immunity. Preclinical findings have proposed TLR9 activation of PDCs by intratumoral injection of CpG-A oligonucleotides (46,57,58). Numerous clinical trials (Clinicaltrials.gov study identifiers: NCT02644967, NCT03445533, NCT03052205, NCT02521870, NCT03084640, and NCT02668770) are ongoing with different compounds being tested, some in association with checkpoint inhibitors (59).…”

Beneficial modulation of the tumor microenvironment and generation of anti-tumor responses by TLR9 agonist lefitolimod alone and in combination with checkpoint inhibitors

“…Secretion of IP-10 depends on the presence of pDC and IFN-alpha. 8,18 The presence of a T cell-inflamed TME in so-called "hot tumors" is linked with improved responses to cancer immunotherapies including checkpoint inhibitors. 30,31 However, MSS cancers are characterized by a lower infiltration of T cells and are known to be poorly responsive to CPI 32,33 rendering it as an ideal model for immunotherapeutic approaches to modulate the TME and enhance the effect of CPI.…”

“…36 However, it should be considered that PTO-modified TLR9 agonists induce a different cytokine pattern in vitro. 8,18 The described immunomodulatory effects of lefitolimod, the potent synergistic anti-tumor responses observed in combination with CPI in murine models, during which no signs of toxicological effects have been observed, and the absence of toxic effects in more than 450 patients in clinical trials [10][11][12][13]37 renders lefitolimod as an ideal combination partner for immunotherapeutic approaches in humans. In fact, a clinical trial in melanoma patients with a combination of lefitolimod and ipilimumab is ongoing and has shown encouraging first data on increase of tumor-infiltrating lymphocytes and a favorable safety profile, and no dose-limiting toxicities have been encountered at any dose level of lefitolimod together with ipilimumab.…”

“…8,9,17 This mode-ofaction (MoA) was confirmed by (i) use of lefitolimod variants devoid of CG-motifs and therefore unable to recognize TLR9, (ii) using human PBMC depleted of TLR9-positive pDC and (iii) blocking of the type I interferon pathway. 8,18 Immune activation by lefitolimod was also confirmed in mice and cynomolgus monkeys.…”

Immunological effects of different types of synthetic CpG oligodeoxynucleotides on porcine cells

“…Almost no information is available for the relationship between the effects of CpG ODNs on the native immune system and the different ODN structures in livestock or domestic animals such as cattle, pigs, horses and poultry. 30 CpG ODNs possess the capacity to enhance innate and adaptive immune responses; thus, we regard CpG ODNs as a promising component in adjuvants for veterinary vaccines in animal husbandry. On the basis of previous research, we know that the ATCGAT hexamer at the core of ODNs is optimal for stimulating porcine PBMCs, and the palindrome created by this hexamer is critical to ODN activity.…”