Degradable Hydrogel for Sustained Localized Delivery of Anti-Tumor Drugs.

Disadvantages of systemically administered immunomodulatory anti-tumor therapies include poor efficacy and high toxicity. Direct intratumoral injection of a drug is often associated with rapid efflux from the site of administration, thus reducing local exposure and therapeutic efficacy, while potentially increasing systemic adverse events. To address this, a sustained release prodrug technology was developed using a transient conjugation (TransConTM) technology to provide long-term high local drug exposure after injection in the tumor while minimizing systemic exposure. TransCon technology for systemic delivery is clinically validated, with multiple compounds in late-stage clinical development and approval of a once-weekly growth hormone for pediatric growth hormone deficiency. As a further application of this technology, this report describes the design, preparation, and functional characterization of hydrogel microspheres as insoluble, yet degradable carrier system. Microspheres were obtained after reaction of PEG-based polyamine dendrimers and bifunctional crosslinkers. Resiquimod, a TLR7/8 agonist, and axitinib, a vascular endothelial growth factor tyrosine kinase inhibitor, were chosen as anti-cancer drugs. The drugs were covalently attached to the carrier by linkers, which released the drugs under physiological conditions. Essentially all resiquimod or axitinib was released over weeks before physical degradation of the hydrogel microsphere was observed. In summary, TransCon Hydrogel technology allows localized sustained-release drug delivery for cancer therapy enabling high local drug concentrations while at the same time ensuring low systemic drug exposure over weeks with a single injection, which may improve the therapeutic index and improve efficacy, while minimizing systemic adverse events. A hydrogel prodrug of resiquimod, TransCon TLR7/8 agonist, is currently being investigated in clinical trials of patients with solid tumors (NCT04799054).

Intratumoral delivery of TransCon™ TLR7/8 Agonist promotes sustained anti-tumor activity and local immune cell activation while minimizing systemic cytokine induction.

BACKGROUND: Intratumoral (IT) delivery of toll-like receptor (TLR) agonists has shown encouraging anti-tumor benefit in preclinical and early clinical studies. However, IT delivery of TLR agonists may lead to rapid effusion from the tumor microenvironment (TME), potentially limiting the duration of local inflammation and increasing the risk of systemic adverse events. METHODS: To address these limitations, TransCon™ TLR7/8 Agonist-an investigational sustained-release prodrug of resiquimod that uses a TransCon linker and hydrogel technology to achieve sustained and predictable IT release of resiquimod-was developed. TransCon TLR7/8 Agonist was characterized for resiquimod release in vitro and in vivo, in mice and rats, and was assessed for anti-tumor efficacy and pharmacodynamic activity in mice. RESULTS: Following a single IT dose, TransCon TLR7/8 Agonist mediated potent tumor growth inhibition which was associated with sustained resiquimod release over several weeks with minimal induction of systemic cytokines. TransCon TLR7/8 Agonist monotherapy promoted activation of antigen-presenting cells in the TME and tumor-draining lymph nodes, with evidence of activation and expansion of CD8+ T cells in the tumor-draining lymph node and TME. Combination of TransCon TLR7/8 Agonist with systemic immunotherapy further promoted anti-tumor activity in TransCon TLR7/8 Agonist-treated tumors. In a bilateral tumor setting, combination of TransCon TLR7/8 Agonist with systemic IL-2 potentiated tumor growth inhibition in both injected and non-injected tumors and conferred protection against tumor rechallenge following complete regressions. CONCLUSIONS: Our findings show that a single dose of TransCon TLR7/8 Agonist can mediate sustained local release of resiquimod in the TME and promote potent anti-tumor effects as monotherapy and in combination with systemic immunotherapy, supporting TransCon TLR7/8 Agonist as a novel intratumoral TLR agonist for cancer therapy. A clinical trial to evaluate the safety and efficacy of TransCon TLR7/8 Agonist, as monotherapy and in combination with pembrolizumab, in cancer patients is currently ongoing (transcendIT-101; NCT04799054).

Site-specific DNA labeling by Staudinger ligation.

Site-specific and chemoselective labeling of DNA is still a difficult task. The Staudinger ligation is a bioorthogonal reaction between azides and phosphines that requires no catalyst to proceed, allowing for mild reaction conditions. The reaction may be extended for site-specific labeling of DNA using azido-modified triphosphates, which can be incorporated site-specifically into DNA strands by DNA polymerases in a template-dependent manner. The azido-modified DNA, in turn, can be labeled by suitable phosphines. This protocol describes (1) the synthesis of an azido-TTP analogue; (2) the enzymatic synthesis of azido-modified DNA; (3) the synthesis of suitable phosphine labels; and (4) the labeling of azido-DNA with biotin-phosphine by Staudinger ligation with approximately 70% conversion.

Novel strategies for the site-specific covalent labelling of nucleic acids.

To broaden the scope of applications in DNA nano- and biotechnology, material science, diagnostics and molecular recognition the functionalization of DNA is of utmost importance. In the last decade many new methods have been developed to achieve this goal. Apart from the direct chemical synthesis of modified DNA by automated phosphoramidite chemistry incorporation of labelled triphosphates and the post-synthetic labelling approach evolved as valuable methods. New bioorthogonal reactions as Diels-Alder, click and Staudinger ligations pushed forward the post-synthetic approach as new insights into DNA polymerase substrate specificity allowed generation and amplification of labelled DNA strands. These novel developments are summarized herein.

DNA conjugation by Staudinger ligation.

Two 5 modified 2'-deoxyuridin triphosphates and a 7 modified 2'-deoxy-7-deazaadenosine were synthesized carrying a terminal azide linked to the base. For probing the sterical influence on incorporation and Staudinger ligation different sized flexible linkers were chosen. All three nucleotides can completely replace their natural counterparts in primer extension as well as polymerase chain reactions (PCR) using Pwo DNA polymerase. For azide labeled primer extension products subsequent conjugation of suitably functionalized phosphines via Staudinger ligation was achieved, e.g. for the conjugation of biotin as an affinity tag.

A nucleoside triphosphate for site-specific labelling of DNA by the Staudinger ligation.

A novel nucleotide building block for enzymatic synthesis of azide modified DNA and subsequent conjugation via Staudinger ligation was developed.