Traditional antibody-drug conjugate (ADC) technology has employed tumor-targeting antibodies to selectively deliver ultrapotent cytotoxins to tumor tissue. While this technology has been highly successful, resulting in the FDA approval of over 10 ADCs, the field continues to struggle with modest efficacy and significant off-target toxicity. Concurrent with the struggles of the ADC field, a new generation of immune-activating therapeutics has arisen, most clearly exemplified by the PD-1/PD-L1 inhibitors that are now part of standard-of-care treatment regimens for a variety of cancers. The success of these immuno-oncology therapeutic agents has prompted the investigation of a variety of new immuno-stimulant approaches, including toll-like receptor (TLR) activators. Herein, we describe the optimization of ADC technology for the selective delivery of a potent series of TLR7 agonists. A series of imidazole[4,5-c]quinoline agonists (as exemplified by compound 1) were shown to selectively agonize the human and mouse TLR7 receptor at low nanomolar concentrations, resulting in the release of IFNα from human peripheral blood mononuclear cells (hPBMCs) and the upregulation of CD86 on antigen-presenting cells. Compound 1 was attached to a deglycosylated (Fc-γ null) HER2-targeting antibody via a cleavable linker, resulting in an ADC (anti-HER2_vc-1) that potently and selectively activated the TLR7 pathway in tumor-associated macrophages via a “bystander” mechanism. We demonstrated that this ADC rapidly released the TLR7 agonist into the media when incubated with HER2+ cells. This release was not observed upon incubation with an isotype control ADC and furthermore was suppressed by co-administration of the naked antibody. In co-culture experiments with HER2+ HCC1954 cells, this ADC induced the activation of the NFκB pathway in mouse macrophages and the release of IFNα from hPBMCs, while a corresponding isotype control ADC did not. Finally, we demonstrated that IP administration of anti-HER2_vc-1 induced complete tumor regression in an HCC1954 xenograft study in SCID beige mice. Unlike related ADC technology that has been reported recently, our technology relies on the passive diffusion of the TLR7 agonist into tumor-associated macrophages rather than Fc-γ-mediated uptake. Based on these observations, we believe that this ADC technology holds significant potential for both oncology and infectious disease applications.
Over the past two decades, antibody drug conjugates (ADCs) and small molecule drug conjugates (SMDCs) have widely employed valine-citruline and related cathepsin-cleavable linkers due to their stability in plasma and their rapid cleavage by lysosomal cathepsins. However, a number of recent studies have illustrated that these linkers are subject to cleavage by exogenous enzymes such as Ces1C and neutrophil elastase, thus resulting in off-target release of drug. As such, there is a need to diversify the portfolio of ADC linkers in order to overcome nonspecific drug release. Rather than targeting cathepsins, we began with an “enzyme agnostic” screen in which a panel of 75 peptide FRET pairs were screened for cleavage in lysosomal extracts and in plasma. Unexpectedly, a series of Asn-containing peptides emerged from this screen as being cleaved far more quickly than traditional ValCit-type linkers while retaining excellent stability in plasma. Catabolism studies demonstrated that these linkers were cleaved by legumain, an asparaginyl endopeptidase that is overexpressed in a variety of cancers and is known to be present in the lysosome. MMAE-containing ADCs that incorporated these new linkers were shown to exhibit highly potent and selective cytotoxicity, comparable to analogous ValCit ADCs. Importantly, the Asn-containing linkers were shown to be completely stable to human neutrophil elastase, an enzyme thought to be responsible for the neutropenia and thrombocytopenia associated with ValCitPABC-MMAE ADCs. The legumain-cleavable ADCs were shown to have excellent stability in both mouse and human serum, retaining >85% of the drug after 1 week of incubation. Moreover, the corresponding small molecule FRET pairs exhibited <10% cleavage after 18 h in mouse and human serum. On the basis of these results, we believe that these new linkers (AsnAsn in particular) have significant potential in both ADC and SMDC drug delivery applications.
In spite of tremendous advances in the design of ADC linkers, there continue to be limited options for the release of payloads that do not contain a free amino group. Herein, we review a variety of cleavable linker strategies that have been employed for both phenolic and aliphatic alcohols. We highlight the advantages and shortcomings of each approach, particularly focusing on technology that has advanced into the clinic and those approaches that are broadly applicable to structurally diverse alcohol-containing payloads.
KRAS is a well-validated anti-cancer therapeutic target, whose transcriptional downregulation has been demonstrated to be lethal to tumor cells with aberrant KRAS signaling. G-quadruplexes (G4s) are non-canonical nucleic acid structures that mediate central dogmatic events, such as DNA repair, telomere elongation, transcription and splicing events. G4s are attractive drug targets, as they are more globular than B-DNA, enabling more selective gene interactions. Moreover, their genomic prevalence is increased in oncogenic promoters, their formation is increased in human cancers, and they can be modulated with small molecules or targeted nucleic acids. The putative formation of multiple G4s has been described in the literature, but compounds with selectivity among these structures have not yet been able to distinguish between the biological contribution of the predominant structures. Using cell free screening techniques, synthesis of novel indoloquinoline compounds and cellular models of KRAS-dependent cancer cells, we describe compounds that choose between KRAS promoter G4near and G4mid, correlate compound cytotoxic activity with KRAS regulation, and highlight G4mid as the lead molecular non-canonical structure for further targeting efforts.
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