Near-infrared duocarmycin photorelease from a Treg-targeted antibody-drug conjugate improves efficacy of PD-1 blockade in syngeneic murine tumor models.

Regulatory T cells (Tregs) play a crucial role in mediating immunosuppression in the tumor microenvironment. Furthermore, Tregs contribute to the lack of efficacy and hyperprogressive disease upon Programmed cell death protein 1 (PD-1) blockade immunotherapy. Thus, Tregs are considered a promising therapeutic target, especially when combined with PD-1 blockade. However, systemic depletion of Tregs causes severe autoimmune adverse events, which poses a serious challenge to Treg-directed therapy. Here, we developed a novel treatment to locally and predominantly damage Tregs by near-infrared duocarmycin photorelease (NIR-DPR). In this technology, we prepared anti-CD25 F(ab')2 conjugates, which site-specifically uncage duocarmycin in CD25-expressing cells upon exposure to NIR light. In vitro, CD25-targeted NIR-DPR significantly increased apoptosis of CD25-expressing HT2-A5E cells. When tumors were irradiated with NIR light in vivo, intratumoral CD25+ Treg populations decreased and Ki-67 and Interleukin-10 expression was suppressed, indicating impaired functioning of intratumoral CD25+ Tregs. CD25-targeted NIR-DPR suppressed tumor growth and improved survival in syngeneic murine tumor models. Of note, CD25-targeted NIR-DPR synergistically enhanced the efficacy of PD-1 blockade, especially in tumors with higher CD8+/Treg PD-1 ratios. Furthermore, the combination therapy induced significant anti-cancer immunity including maturation of dendritic cells, extensive intratumoral infiltration of cytotoxic CD8+ T cells, and increased differentiation into CD8+ memory T cells. Altogether, CD25-targeted NIR-DPR locally and predominantly targets Tregs in the tumor microenvironment and synergistically improves the efficacy of PD-1 blockade, suggesting that this combination therapy can be a rational anti-cancer combination immunotherapy.

SNAP-Tag-Targeted MRI-Fluorescent Multimodal Probes.

Magnetic resonance imaging (MRI) is a powerful imaging modality, widely employed in research and clinical settings. However, MRI images suffer from low signals and a lack of target specificity. We aimed to develop a multimodal imaging probe to detect targeted cells by MRI and fluorescence microscopy. We synthesized a trifunctional imaging probe consisting of a SNAP-tag substrate for irreversible and specific labelling of cells, cyanine dyes for bright fluorescence, and a chelated GdIII molecule for enhancing MRI contrast. Our probes exhibit specific and efficient labelling of genetically defined cells (expressing SNAP-tag at their membrane), bright fluorescence and MRI signal. Our synthetic approach provides a versatile platform for the production of multimodal imaging probes, particularly for light microscopy and MRI.

Antibody guided activatable NIR photosensitizing system for fluorescently monitored photodynamic therapy with reduced side effects.

Photodynamic therapy (PDT) utilizing an organic dye (photosensitizer) capable of killing cancer cells in the body upon light irradiation is one of the promising non-invasive treatment modalities for many cancers. A known drawback of PDT is a side-effect caused by existing photosensitizers to organs due to insufficient specificity and accidental light exposure of a patient during the delivery of the photosensitizer in the bloodstream. To overcome this issue, we developed a novel antibody guided, activatable photosensitizing system, Ab-mI2XCy-Ac, where the trastuzumab (Ab) is linked to the non-active (not phototoxic and not fluorescent) dye, mI2XCy-Ac, that contains the hydroxyl group protected by acetyl (Ac). This targeting, non-photo-active conjugate was shown to be safely (without detectable side-effects) delivered to the targeted tumor, where it is activated by the esterase-mediated acetyl group cleavage and effectively treats the tumor upon NIR light irradiation. It was demonstrated in the Her2 positive BT-474 tumor mouse model that the treatment efficacy of the activatable photosensitizing system is about the same as for the permanently active photosensitizer, Ab-mI2XCy, while the side-effects are noticeably reduced. In addition, this activatable system enables fluorescence monitoring of the photosensitizer activation events.

A novel, dual action chimera comprising DNA methylating agent and near-IR xanthene-cyanine photosensitizer for combined anticancer therapy.

A facile synthesis, biological evaluation and photodynamic properties of novel activatable anticancer molecular hybrids (chimeras) Ch and I-Ch are described. The chimeras consist of DNA methylating methyl triazene moiety and fluorogenic xanthene-cyanine (XCy) or iodinated xanthene-cyanine (I-XCy) photosensitizer. These two anticancer core structures are bound by means of a self-immolative 4-aminobenzyl alcohol linker. The hydrolytic cleavage of the carbamate protecting group promotes activation of both DNA methylating monomethyl triazene and phototoxic xanthene-cyanine dye providing, in addition, a near-IR emission signal for detection of the drug activation events. Preliminary antiproliferative assay demonstrates that the developed chimeras exhibit higher antitumor activity in the breast cancer cell line upon near-IR light irradiation compared to their structural constituents, xanthene-cyanine photosensitizer and monomethyl triazene substance.

Targeted methylation facilitates DNA double strand breaks and enhances cancer suppression: A DNA intercalating/methylating dual-action chimera Amonafidazene.

A DNA intercalating agent Amonafide interferes with topoisomerase 2 (Topo II) activity and prevents re-ligation of DNA strands, leading to double strand breaks (DSB). If DSB repair fails, cells stop dividing and eventually die. In a search of approaches to enhance anti-cancer activities of Topo II inhibitors, we hypothesized that introduction of additional damage in proximity to the DSB may suppress DNA repair and enhance cancer cell killing. Accordingly, chimeric molecules were created that target a DNA alkylating component to the proximity of Topo II-induced DSBs. These chimeras consist of Amonafide or its 4-amino isomer, and DNA methylating methyl triazene moiety Azene protected with a carbamate group, connected via linker. Treatment of cancer cells with the chimeric molecules leads to significantly higher number of DSBs, which were repaired slower compared to Amonafide or monomethyl triazene-treated cells. On the other hand, methyl triazene linked to non-intercalating Amonafide analogs was ineffective. Together, these data strongly support our hypothesis. In line with increased DSBs, the chimeric molecules exhibited significantly higher antiproliferative activity in cancer cell lines compared to Amonafide or monomethyl triazene constituent Azene. We utilized the fluorescent properties of chimera Amonafidazene to develop ''photo-switchable'' reporting system to monitor the prodrug activation. Using this approach, we found that the chimera accumulated and was activated at the tumor sites specifically and demonstrated significantly stronger tumor suppressing activities compared to Amonafide in a xenograft model. Therefore, targeting alkylating groups to the proximity of DSB sites may become an effective approach towards enhancing anti-cancer activities of inhibitors of topoisomerases.

Ratiometric Fluorescence Monitoring of Antibody-Guided Drug Delivery to Cancer Cells.

Ratiometric measurements utilizing two independent fluorescence signals from a dual-dye molecular system help to improve the detection sensitivity and quantification of many analytical, bioanalytical, and pharmaceutical assays, including drug delivery monitoring. Nevertheless, these dual-dye conjugates have never been utilized for ratiometric monitoring of antibody (Ab)-guided targeted drug delivery (TDD). Here, we report for the first time on the new, dual-dye TDD system, Cy5s-Ab-Flu-Aza, comprising the switchable fluorescein-based dye (Flu) linked to the anticancer drug azatoxin (Aza), reference pentamethine cyanine dye (Cy5s), and Her2-specific humanized monoclonal Trastuzumab (Herceptin) antibody. The ability of ratiometric fluorescence monitoring of drug release was demonstrated with this model system in vitro in the example of the human breast cancer SKBR3 cell line overexpressing Her2 receptors. The proposed approach for designing ratiometric, antibody-guided TDD systems, where a "drug-switchable dye" conjugate and a reference dye are independently linked to an antibody, can be expanded to other drugs, dyes, and antibodies. Replacement of the green-emitting dye Flu, which was found not detectable in vivo, with a longer-wavelength (red or near-IR) switchable fluorophore should enable quantification of drug release in the body.

Quantification of Drug Release Degree In Vivo Using Antibody-Guided, Dual-NIR-Dye Ratiometric System.

Fluorescent dyes linked to drug delivery systems provide important real-time information on the efficacy of drug delivery. However, the quantitative monitoring of drug distribution is challenging because of interferences from the biological sample and instrumental setup. To improve quantification of anticancer drug delivery followed by drug release in tumor, we equipped an antibody-drug conjugate (ADC) with a turn-on near-infrared (NIR) dye, sensitive to drug release, and a reference NIR dye. In this study, chlorambucil (CLB) was chosen as a model anticancer drug and Trastuzumab monoclonal antibody specific to Her2 receptors overexpressed in many tumors was taken as the carrier. The advantage of the obtained dual-dye ratiometric system for drug release monitoring was demonstrated in mice model.