TLR Agonists Emerge as Potential New Partner for Checkpoint Inhibitors

These first responders of the immune system help lay the groundwork for optimal activation of the adaptive immune response. Thus far, TLR agonists have had limited clinical success, partly due to the seemingly duplicitous role that many TLRs play in cancer.

Development efforts are far from over, however, and a greater understanding of the complexities of TLR signaling in cancer, combined with novel drug design, has yielded a growing number of investigational drugs. Particularly noteworthy are the new generation of TLR9 agonists, whose use could help to establish an ideal backdrop for optimal activation of T cell—based immunotherapies. Ongoing clinical trials have demonstrated exciting activity for TLR9 agonists combined with immune checkpoint inhibitors (ICIs).

Frontline Defenses

The innate immune response is the human body’s first line of defense against invading pathogens. Its sentries—macrophages, neutrophils, natural killer (NK) cells, and dendritic cells (DCs)—patrol the body armed with pattern recognition receptors (PRRs). These germline-encoded receptors recognize unique molecular patterns, known as pathogen-associated molecular patterns (PAMPs). To maintain cellular homeostasis, PRRs can also bind to danger-associated molecular patterns (DAMPs), potentially harmful “self” material released by dead or dying cells.

The best-known PRRs are TLRs, a group of 10 related proteins first discovered in the 1990s. Their importance to innate immunity was underscored by the awarding of the 2011 Nobel Prize in Physiology or Medicine to the investigators who uncovered their role.^1-3

The specific TLR that is activated depends on the nature of the encountered threat; TLR9, for example, recognizes unmethylated nucleotide couplets, CpGs, in which a cytosine is followed by a guanosine nucleotide. A hallmark of microbial DNA, CpGs occur comparatively less often in human DNA, where they represent approximately 1% of the genome and tend to be methylated.

The cellular localization of a given TLR family member reflects where its activating PAMP/DAMP ligand is found: TLR1, 2, 5, and 6 are located on the cell surface, where they respond primarily to bacterial proteins; TLR3, 7, 8, and 9 are found on intracellular endosomal membranes because they are activated by foreign nucleic acids that are taken up into the cell during infection; and TLR4 and 10 have been described in both locations^3-6 (Figure⁵).

TLRs consist of an extracellular domain that protrudes from the external surface of the membrane and binds to the PAMP/DAMP, a transmembrane domain, and an intracellular portion that coordinates downstream signaling pathways via a conserved toll/IL-1 receptor (TIR) domain.

Via the TIR domain, TLRs recruit adaptor proteins, the best known of which is MyD88. Downstream, a number of signaling pathways are activated, ultimately upregulating key transcription factors (eg, NF-κB) that in turn govern the expression of genes encoding proinflammatory cytokines that are integral to the innate immune response.

Figure. The Toll-Like Receptor Family in Action

TLR signaling is also involved in tissue homeostasis via wound healing and tissue repair/regeneration; the seemingly contradictory functions of cell survival and apoptosis (see below); and promotion of the antigen-specific and longer-lasting adaptive immune response through activation and maturation of DCs and NK cells, enhanced antigen presentation, and induction of T helper and cytotoxic T cells.^3-6

A Moving Target

The ability of the innate immune response to recognize components of damaged and dying cells has implicated it in the generation of anti-tumor immunity. Chromosomal and DNA damage is a cancer hallmark resulting from the genomic instability inherent to many tumor types and can produce an abundant source of DAMPs.

As central mediators of innate immunity, TLRs have emerged as a therapeutic target. A range of TLR agonists, designed to boost TLR activity, have been developed. To date, only a handful of TLR agonists have been approved by the FDA for use in patients with cancer.

Bacille Calmette-Guérin, a live attenuated Mycobacterium bovis preparation, is a TLR2 and 4 agonist and is approved for intravesical use in nonmuscle invasive bladder cancer following tumor resection. Imiquimod, a TLR7 agonist, is approved as a topical cream for superficial basal cell carcinoma. Monophosphoryl lipid A (a derivative of lipid A, the biologically active part of lipopolysaccharide) is a TLR4 agonist approved as an adjuvant in the human papillomavirus vaccine for cervical cancer prevention.

A major obstacle to the development of TLR agonists is the complex role of TLR signaling in cancer development and progression. Increasing evidence has demonstrated that TLRs are often overexpressed by cancer cells and cells of the tumor microenvironment.

Although TLRs undoubtedly have antitumorigenic effects, they can also promote oncogenesis and foster a proinflammatory microenvironment that is conducive to tumor growth and survival. The mechanisms underlying these contrary roles are not yet fully understood but have become a hot-button topic in cancer research. Current evidence indicates that the pro- or antioncogenic potential of a TLR depends on contextual factors such as the cell type and TLR subclass.^3-8

Fresh Excitement

As investigators continue to unravel the complexities, TLRs have remained a promising target and a growing number of agents are in clinical development (Table). The largest body of evidence for antitumor activity involves TLR9 agonists, and this is where clinical development efforts have been focused.

TLR9 agonists are composed of short, synthetic stretches of single-stranded DNA containing a CpG dinucleotide motif, known as CpG oligodeoxynucleotides (CpG-ODNs), and are designed to mimic the PAMP recognized by TLR9.^9,10 They are predominantly designed for local delivery—intratumoral and subcutaneous— largely because of previous study findings demonstrating that local administration yields higher antitumor effects and limits systemic toxicity.^11,12 Initial clinical trials were disappointing but more recently, a new generation of CpG-ODNs with unique features designed to improve their efficacy have been developed.¹³

This class of drugs has been dubbed immune surveillance reactivators because of their potential capacity to not only augment innate antitumor immunity but also promote antigen-specific T- and B-cell responses and thus boost adaptive immunity.^13-15

Lefitolimod

The leading candidate in the field is lefitolimod (MGN1703), which has been investigated as both a vaccine adjuvant and a therapeutic drug in its own right.¹⁶ In a phase I trial of 28 patients with metastatic solid tumors, lefitolimod was safe and well tolerated, with antitumor efficacy in heavily pretreated patients.¹⁷

The phase II IMPACT trial evaluated lefitolimod as maintenance therapy in patients with metastatic colorectal cancer (CRC). In results published in 2014, lefitolimod demonstrated disease control compared with placebo and was well tolerated.¹⁶ A phase III trial, IMPALA, is ongoing in this setting, and the results are expected sometime this year.¹⁸

In another phase II study, lefitolimod was used as maintenance therapy in patients with extensive-stage small cell lung cancer. A total of 103 patients who experienced an objective response following 4 cycles of platinum-based frontline induction therapy were randomized to receive lefitolimod (n = 62) or standard of care (n = 41). Although no effect on overall survival (OS) was observed in the overall population, lefitolimod had a favorable OS impact in those with a low frequency of activated CD86-positive B cells and those with chronic obstructive pulmonary disease.¹⁵

As immune surveillance reactivators, agonists of TLR9 and other TLRs could provide the ideal backdrop for optimal activity of immunotherapeutics that rely on activated T cells infiltrating the tumor microenvironment, such as ICIs. A preclinical study of lefitolimod in a CRC model, presented at the 2018 American Society of Clinical Oncology (ASCO) Annual Meeting, highlighted the significant potential for synergy between TLR9 agonists and ICIs. Intratumoral injection of lefitolimod resulted in reduced tumor growth, increased infiltration of activated T cells, and recruitment of macrophages.¹⁴

SD-101

A plethora of ongoing clinical trials are evaluating combinations of TLR agonists and ICIs. Another promising TLR9 agonist, SD-101, is showing activity in this context.

Two recent presentations reported results from the ongoing phase II trial SYNERGY-001/ KEYNOTE-184, in which SD-101 is being administered in combination with pembrolizumab (Keytruda), a PD-1 inhibitor. Among 23 patients with metastatic squamous cell carcinoma of the head and neck who had not received a prior PD-1/ PD-L1 inhibitor, the overall response rate (ORR) was 30.4%, including 7 partial responses (PRs).¹⁹

Meanwhile, in patients with treatment-naïve, unresectable melanoma, the ORR was 70%, including 5 complete responses (CRs) and 28 PRs among those 47 participants treated with 2-mg intratumoral SD-101 and 48%, including 2 CRs and 17 PRs, for 40 of those treated with an 8-mg dose. The most frequent grade ≥3 TRAEs were headache, myalgia, malaise, fatigue, chills, and injection-site pain.²⁰ Additional results are scheduled to be presented at the 2019 ASCO Annual Meeting in June.²¹

Tilsotolimod and CMP-001

Two additional TLR9 agonists, tilsotolimod (IMO2125) and CMP-001, are also showing promise in combination with ICIs. In an ongoing phase II study, 21 patients with metastatic melanoma refractory to PD-1 inhibitors were treated with the combination of tilsotolimod (8 mg administered into a single tumor lesion) in combination with ipilimumab (Yervoy), a CTLA-4 ICI. The ORR was 38.1%, including 2 CRs and 6 PRs, and the most common immune-related AEs were hypophysitis, hepatitis, gastritis, colitis, adrenal insufficiency, and Guillain-Barré syndrome.²² Last year, the phase III ILLUMINATE-301 trial was launched to evaluate this combination.²³

The ongoing phase IB CMP-001-001 trial is testing the combination of CMP-001 and pembrolizumab in patients with advanced melanoma resistant to PD-1 inhibition.

Table. Select Ongoing Clinical Trials of TLR Inhibitors

Findings reported for 53 patients treated as of December 2017 demonstrated ORRs of 22.5% among 40 patients dosed weekly and 7.7% among 13 patients treated every 3 weeks. TRAEs included fever, headache, nausea and vomiting, hypotension, and rigors.²⁴

References

1. The Nobel Prize in Physiology or Medicine 2011 [press release]. Stockholm, Sweden: The Nobel Assembly at Karolinska Institute; October 10, 2011. nobelprize.org/prizes/medicine/2011/press-release/. Accessed April 25, 2019.
2. Vogel SN. How discovery of Toll-mediated innate immunity in drosophila impacted our understanding of tlr signaling (and vice versa). J Immunol. 2012;188(11):5207-5209. doi: 10.4049/jimmunol.1201050.
3. Braunstein MJ, Kucharczyk J, Adams S. Targeting Toll-like receptors for cancer therapy. Targeted Oncology. 2018;13(5):583-598. doi: 10.1007/s11523-018-0589-7.
4. Du B, Jiang Q, Cleveland J, Liu B, Zhang D. Targeting Toll-like receptors against cancer. J Cancer Metastasis Treat. 2016;2:463-470. doi: 10.20517/2394-4722.2016.62.
5. Li K, Qu S, Chen X, Wu Q, Shi M. Promising targets for cancer immunotherapy: TLRs, RLRs, and STING-mediated innate immune pathways. Int J Mol Sci. 2017;18(2):404. doi: 10.3390/ijms18020404.
6. Liu C, Han C, Liu J. The Role of Toll-like Receptors in Oncotherapy. Oncol Res. 2019doi: 10.3727/096504019x15498329881440.
7. Gonzalez-Cao M, Karachaliou N, Santarpia M, Viteri S, Meyerhans A, Rosell R. Activation of viral defense signaling in cancer. Ther Adv Med Oncol. 2018;10:1758835918793105. doi: 10.1177/1758835918793105.
8. Shi M, Chen X, Ye K, Yao Y, Li Y. Application potential of toll-like receptors in cancer immunotherapy: systematic review. Medicine (Baltimore). 2016;95(25):e3951. doi: 10.1097/md.0000000000003951.
9. TLR9 agonists: double-edge sword for immune therapies. Invivogen website. invivogen.com/review-tlr9-agonists. Accessed May 2, 2019.
10. Melisi D, Frizziero M, Tamburrino A, et al. Toll-like receptor 9 agonists for cancer therapy. Biomedicines. 2014;2(3):211-228. doi: 10.3390/biomedicines2030211.
11. Engel AL, Holt GE, Lu H. The pharmacokinetics of Toll-like receptor agonists and the impact on the immune system. Expert review of clinical pharmacology. 2011;4(2):275-289. doi: 10.1586/ecp.11.5.
12. Ito H. Combination therapy with TLR7 agonist and radiation is effective for the treatment of solid cancer. Ann Transl Med. 2016;4(5):95. doi: 10.21037/atm.2015.12.49.
13. Wittig B, Schmidt M, Scheithauer W, Schmoll H-J. MGN1703, an immunomodulator and toll-like receptor 9 (TLR-9) agonist: From bench to bedside. Crit Rev Oncol Hematol. 2015;94(1):31-44. doi: 10.1016/j.critrevonc.2014.12.002.
14. Schmidt M, Oswald D, Volz B, Wittig B, Kapp K. Modulation of T cell and macrophage tumor infiltration by the TLR9 agonist lefitolimod in a murine model of colorectal cancer. J Clin Oncol. 2018;36(suppl 4):687. doi: 10.1200/JCO.2018.36.4_suppl.687.
15. Thomas M, Ponce Aix S, Navarro Mendivil A, et al. Maintenance treatment with the TLR9 agonist lefitolimod in extensive-stage small-cell lung cancer (ES-SCLC): final results from the randomized phase II IMPULSE study. Ann Oncol. 2019;29(suppl 8):viii596-viii602. doi: 10.1093/annonc/mdy298.
16. Schmoll HJ, Wittig B, Arnold D, et al. Maintenance treatment with the immunomodulator MGN1703, a Toll-like receptor 9 (TLR9) agonist, in patients with metastatic colorectal carcinoma and disease control after chemotherapy: a randomised, double-blind, placebo-controlled trial. J Cancer Res Clin Oncol. 2014;140(9):1615-1624. doi: 10.1007/s00432-014-1682-7.
17. Weihrauch MR, Richly H, von Bergwelt-Baildon MS, et al. Phase I clinical study of the toll-like receptor 9 agonist MGN1703 in patients with metastatic solid tumours. Eur J Cancer. 2015;51(2):146-156. doi: 10.1016/j.ejca.2014.11.002.
18. Factsheet: Lefitolimod. Mologen AG website. mologen.com/sites/mologen-ag/files/Investor%20Kit/German/190207_MGN%20Factsheet%20Lefitolimod.pdf. Accessed May 2, 2019.
19. Cohen EE, Algazi A, Laux D, et al. Phase 1b/2, open label, multicenter study of intratumoral SD-101 in combination with pembrolizumab in anti-PD-1 treatment naive patients with recurrent or metastatic head and neck squamous cell carcinoma (HNSCC). Ann Oncol. 2018;29(suppl 8):viii372-viii399. doi: 10.1093/annonc/mdy287.
20. Long GV, Milhem M, Amin A, et al. Phase 1b/2, open label, multicenter, study of the combination of SD-101 and pembrolizumab in patients with advanced melanoma who are naïve to anti-PD-1 therapy. Ann Oncol. 2018;29(suppl 8): mdy424.055. doi: 10.1093/annonc/mdy424.055.
21. Dynavax to present data on Toll-like receptor 9 agonist SD-101 at the ASCO annual meeting 2019 [press release]. Berkeley, CA: Dynavax Technologies Corporation; April 18, 2019. globenewswire.com/news-release/2019/04/18/1806857/0/en/Dynavax-to-Present-Data-on-Toll-like-Receptor-9-Agonist-SD-101-at-the-ASCO-Annual-Meeting-2019.html. Accessed April 23, 2019.
22. Diab A, Rahimian S, Haymaker CL, et al. A phase 1/2 study to evaluate the safety and efficacy of intratumoral injection of the TLR9 agonist tilsotolimod (IMO-2125) in combination with ipilimumab in patients with PD-1 inhibitor refractory metastatic melanoma. Poster presented at 2018 American Society of Clinical Oncology annual meeting; June 1-5, 2019; Chicago, IL. Abstract 9515. meetinglibrary.asco.org/record/159086/poster.
23. Idera Pharmaceuticals announces launch of global phase 3 trial evaluating IMO-2125 in combination with ipilimumab for the treatment of anti-PD-1 refractory melanoma (ILLUMINATE-301) [press release]. Exton, PA and Cambridge, MA: Idera Pharmaceuticals; March 1, 2018. ir.iderapharma.com/news-releases/news-release-details/idera-pharmaceuticals-announces-launch-global-phase-3-trial. Accessed April 24, 2019.
24. Milhem M, Gonzales R, Medina T, et al. Abstract CT144: Intratumoral toll-like receptor 9 (TLR9) agonist, CMP-001, in combination with pembrolizumab can reverse resistance to PD-1 inhibition in a phase Ib trial in subjects with advanced melanoma. Cancer Res. 2018;78(suppl 13):CT144. doi: 10.1158/1538-7445.Am2018-ct144.

Toll-like receptors (TLRs), a group of proteins that are components of innate immunity, are emerging as promising targets in a new wave of immunotherapies under development.