June 22, 2024

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TLRs agonists: innate immunity to the forefront of immunotherapy?

TLRs agonists: innate immunity to the forefront of immunotherapy?


TLRs agonists: innate immunity to the forefront of immunotherapy?

On December 6, 2021, Bolt Therapeutics announced the clinical data of its HER2-TLR7/8 agonist ADC (BDC-001) at ESMO, ORR 2.5%, DcR 32.5%, and the stock price fell 56% instantly. Might we need to look at TLRs agonists differently?


Pattern recognition receptors (PRRs) are the core molecules for initiating and maintaining innate immunity, mainly including TLRs, RGR family and cGAS-STING. They monitor local infection and/or tissue damage, thereby preventing systemic infection, and monitor malignant cell generation.


Intratumoral immunotherapy using TLRs agonists aims to induce or enhance local tumor inflammation and immunity by mimicking intracellular microorganisms (viruses or bacteria), thereby evoking cytotoxic CD8+ T cell responses, promoting TILs infiltration, and stimulating CD4+ T cells produce effector molecules such as IFN-γ, which are natural companions of PD-1 antibody drugs. Combination therapy is the future. Use as a vaccine adjuvant is also the direction of future development.


TLR-9 agonists

Found in the endosomes of immune cells such as myeloid cells, B cells, and plasmacytoid dendritic cells (pDCs), TLR9 recognizes contiguous CpG nucleotide sequences in bacterial or viral double-stranded DNA (dsDNA) that In contrast to mammalian sequences, it is unmethylated.

TLR9 agonists consist of short, contiguous CpG oligonucleotides with pro-inflammatory activity that can be grouped into three classes based on their structure and the most abundant cytokines they induce:
Type A : mainly induces type I interferon IFNα/β;

Type B : induces TNF, IL-12, IFNγ and IL-6;

Type C : Induction of the above two groups of cytokines, the most suitable for anti-tumor immunity.



The TLR9B-type agonist developed by Pfizer more than 10 years ago has been discontinued due to negative Phase III results.



SD-101 is a TLR9 agonist. Dynavax has previously been clinically studied in melanoma and head and neck squamous cell carcinoma (NCT02521870), lymphoma (NCT02266147), etc.

The published results showed that it increased the number of tumor-infiltrating lymphocytes, showing effective sex.

It is currently being developed by TriSalus Life Sciences and is mainly used in combination with immune checkpoint inhibitors. It is currently in Phase 2 clinical trials for melanoma, head and neck squamous cell carcinoma, and breast cancer.

TLRs agonists: innate immunity to the forefront of immunotherapy?
From TriSalus Life Sciences official website


Tilsotolimod (IMO-2125)

Tilsotolimod, another very promising TLR9 agonist, was developed by Idera Pharmaceuticals and received US FDA Fast Track for the treatment of anti-PD-1 antibody-refractory melanoma (in combination with ipilimumab), as well as Orphan Drug Designation For the treatment of stage IIb-IV melanoma.

Currently in the pivotal Phase 3 melanoma clinical trial (NCT03445533), its Phase I/II ILLUMINATE-204 trial (NCT02644967), with data from patients with refractory melanoma, the objective response rate (ORR) was 22% and the disease control rate was 71% , the median overall survival (OS) time was 21 months.


TLRs agonists: innate immunity to the forefront of immunotherapy?
From the official website of Idera Pharmaceuticals


The TLR9 agonist developed by Mologen AG is currently recruited for clinical trials including HIV and advanced solid tumors.

NCT03837756, Combining TLR9 Agonist With bNAbs for Reservoir Reduction and Immunological Control of HIV (TITAN), Phase2

NCT02668770, Ipilimumab (Immunotherapy) and MGN1703 (TLR Agonist) in Patients With Advanced Solid Malignancies, Phase 1

CMP-001/ vidutolimod

A non-infectious biological virus-like particle, developed by Checkmate Pharmaceuticals, containing CpG-ADNA.

TLRs agonists: innate immunity to the forefront of immunotherapy?From the official website of Checkmate Pharmaceutical


A number of phase 2 clinical trials have been carried out for melanoma, head and neck squamous cell carcinoma, and skin squamous cell carcinoma, and positive results have been achieved in PD-1-resistant patients.



TLR4 agonists

TLR4, a cell surface receptor that recognizes bacterial lipopolysaccharide (LPS), was discovered for its role in septic shock and DC maturation.

In addition to LPS, TLR4 also responds to endogenous stimuli such as the nuclear protein HMGB1. Given that TLR4 is the primary mediator of septic shock, systemic targeting is clearly dangerous; therefore, local targeting is required.


The TLR4 agonist G100 is a fully synthetic analog of LPS. The compound has been tested intratumorally in patients with Merkel cell carcinoma, and objective tumor regression has been observed in both neoadjuvant and metastatic settings.

In a phase I/II trial in patients with follicular lymphoma, G100 was combined with low-dose pembrolizumab or rituximab (NCT02501473); the approach was very safe, with an ORR of 26%.

However, after the original research company Immune Design was acquired by Merck, many clinical trials were withdrawn (such as NCT03742804).


TLR3 agonists

TLR3 is an endosome receptor for dsRNAs that normally constitute viral genomes or intermediates during the viral replication cycle. poly I:C is a self-hybridizing dual sRNA analog that was originally described as a potent inducer of IFNα/β production by a variety of leukocytes.

TLR3 is significantly expressed in dendritic cells, including classical type 1 dendritic cells (cDC1), which are responsible for cross-presenting tumor-associated antigens to CD8+ T cells.

The most studied TLR3 agonists are: AMP-516/Rintatolimod/Ampligen, Hiltonol, BO-112
The majority of published intratumorally administered TLR3 agonists are Hiltonol, which has been used as an adjuvant in several vaccines and induces IFNα/β responses when administered subcutaneously alone in healthy volunteers. Intratumoral injection of Hiltonol alone resulted in tumor control, and in combination with DC vaccine radiation therapy, a small number of patients with metastatic solid tumors showed significant disease control.



AIM ImmunoTech is developed and is currently undergoing Phase 2 clinical trials for ovarian cancer and breast cancer.

TLRs agonists: innate immunity to the forefront of immunotherapy?From the official website of AIM ImmunoTech



Highlight Therapeutics research and development. In addition to TLR3, BO-112 activates the cytoplasmic RGR family RNA helicases MDA-5 and RIG-I65, and possibly PKR (another cytoplasmic PRR of dsRNA). There are several clinical trials currently underway.

NCT04508140 Study of BO-112 With Pembrolizumab for Colorectal or Gastric/GEJ Cancer With Liver Metastasis Phase2

NCT04570332 BO-112 With Pembrolizumab in Unresectable Malignant Melanoma (SPOTLIGHT203) Phase2

NCT04777708 BO-112 and Pembrolizumab for the Treatment of PD-1/PD-L1 Refractory Liver Cancer Phase1

After intratumoral injection, the compound killed some tumor cells through mechanisms such as autophagy.

Repeated intratumoral injections of BO-112 are safe in humans and induce infiltration of type I IFN and CD8+ T cells into the injection site. The addition of BO-122 to the anti-PD-1 mAb was also well tolerated, with objective responses in 3 of 28 patients (11%) with primary resistance to the anti-PD-1 mAb (ref. 5).



TLR7/TLR8 agonists

TLR7 and TLR8 are endosome receptors that recognize single-stranded RNAs (e single-strand RNAs, ssRNAs) with viral characteristics, such as abundant GU dinucleotide motifs, imidazoquinoline (IQ) are agonists of these receptors and are pro-inflammatory stimulating molecules such as imiquimod and resiquimod.

In addition, repeated topical application of Imiquimod cream to superficial basal cell carcinoma (BCC) lesions resulted in a response in the majority of patients that was curative or facilitated subsequent more conservative surgical treatment, which was approved by the FDA in 2004. Infiltration of pDCs is a key effector mechanism, and NK cells, T cells, and IFN may also be involved.

Dutch researchers used the cream to treat vulvar intraepithelial neoplasia, a human papillomavirus (HPV)-related precancerous disease, with a complete response (CR) rate of 80% and was well tolerated.

In addition, Imiquimod has been tested for the treatment of breast cancer skin metastases, increasing the local response to radiation from 11% to 66%. However, intratumoral injection of TLR7/8 agonists remains to be clinically explored.


MEDI9197 (3M-052)

MEDI9197 (3M-052) is a TLR7/8 agonist developed by MedImmune.
In 2015, a clinical study (NCT02556463) of intratumoral injection of a single agent (or in combination with Durvalumab and/or Palliative Radiation) in the treatment of solid tumors was registered, and the strategy was adjusted and terminated in 2018.

It is now used as an adjuvant in vaccines. Two HIV vaccine clinical trial registrations (NCT04177355, NCT04915768)





TLRs are the most well-studied pattern recognition receptors and are the core molecules of innate immune activation. TLRs agonists are an important direction for anti-tumor immunotherapy.

In addition, TLRs agonists activate innate immunity and are the cornerstone of adaptive immune activation, so they have natural properties in combination with PD-1 therapy.

However, the systemic administration of TLRs agonists may lead to systemic inflammation, which is an undesirable treatment-related side effect, so current clinical development focuses on local intratumoral injection to localize inflammation to the tumor.



1. Marabelle, A., Tselikas, L., de Baere, T. & Houot, R. Intratumoral immunotherapy: using the tumor as the remedy. Ann. Oncol. 28, xii33–xii43 (2017).
2. Petitprez, F. et al. B cells are associated with survival and immunotherapy response in sarcoma. Nature 577, 556–560 (2020).
3. Helmink, BA et al. B cells and tertiary lymphoid structures promote immunotherapy response. Nature 577, 549–55.55 (2020)
4. Ribas, A. et al. SD-101 in combination with pembrolizumab in advanced melanoma: results of a phase Ib, multicenter study. Cancer Discov. 8, 1250–1257 (2018)
5. Marquez-Rodas, I. et al. Intratumoral nanoplexed poly I:C BO-112 in combination with systemic anti-PD-1 for patients with anti-PD-1-refractory tumors. Sci. Transl Med. 12, eabb0391 ( 2020)
6. Lilit Karapetyan et al, Toll-Like Receptor 9 Agonists in Cancer, OncoTargets and Therapy 2020:13 10039–10060

TLRs agonists: innate immunity to the forefront of immunotherapy?

(source:internet, reference only)

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