1185 Optimization of purine-based TLR7 agonists as payloads for immune-stimulating antibody conjugates (ISACs)
Bibliographic record
Abstract
Background Immune-stimulating antibody conjugates (ISACs) consist of antibodies conjugated to immune stimulants and are designed to induce antitumor immune response. Despite promising preclinical results, ISAC clinical development has been hampered by systemic toxicities or lack of efficacy. Substituted purines have previously been identified as a privileged scaffold to elicit TLR7 activation. Here, we demonstrate newly designed purine-based TLR7 agonists conjugated to trastuzumab which show significant tumor volume reduction in a HER2-high gastric cancer xenograft model without associated body weight loss (BWL) in healthy mice. Methods A library of TLR7 agonists was generated by varying substituents at C2- and N9-positions of a common 6-amino-8-hydroxy-purine scaffold, and the structure-activity relationship was studied in vitro using human and mouse TLR7 reporter gene assays (RGAs) as well as measuring cytokine secretion from human peripheral blood mononuclear cells (PBMCs) and mouse splenocytes. Lead TLR7 agonists were conjugated to trastuzumab, and the resulting ISACs were evaluated in vitro for their abilities to induce the production of interleukin-6 (IL-6) from human PBMCs or mouse splenocytes co-cultured with NCI-N87 tumor cells. Selected ISACs were tested for efficacy (single iv injection at 2.5 mg/kg) in mice bearing NCI-N87 tumors (figure 1) and for tolerability (single iv injection at 3, 15, and 45 mg/kg) in healthy mice (figure 2). Results We prepared ~220 new TLR7 agonists with different substituents at C2- and N9-positions of the purine scaffold. Compounds with IC50 <100 nM in both human and mouse TLR7 RGAs were further screened for their abilities to induce production of cytokines in PBMCs and mouse splenocytes. Certain substituents were found to be highly immunostimulatory in both human and murine settings. Lead TLR7 agonists were conjugated to trastuzumab with a drug-to-antibody ratio of ~4 using cleavable or non-cleavable linkers. ISACs capable of inducing IL-6 production from PBMCs or splenocytes co-cultured with tumor cells were further tested in an NCI-N87 xenograft model in comparison to unconjugated trastuzumab and trastuzumab conjugated with the same linker-payload as NJH395, a clinical benchmark ISAC. Selected ISACs were tested for tolerability in healthy mice with a lead ISAC (Trastuzumab-MTvkPABC-P5) identified, capable of inducing tumor regression without causing BWL. Conclusions We demonstrated the potential of using novel purine-based TLR7 agonists as payloads for ISACs. In contrast to other TLR7-agonist conjugates, our lead ISAC appears to have a sufficiently wide therapeutic window displaying efficacy in an NCI-N87 xenograft model at 2.5 mg/kg without causing BWL in healthy mice at 45 mg/kg. Ethics Approval All animal studies were performed in accordance with Institutional Animal Care and Use Committee (IACUC)-approved protocols.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.001 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".