Human serum albumin (HSA) is a clinically validated drug carrier that improves drug delivery to tumor tissues. However, clinical imaging strategies are lacking to stratify patients who will benefit from HSA-bound drugs. In this study, we site-selectively radiolabeled HSA with zirconium-89 (89Zr), using the octadentate chelator DFO*, to provide an imaging probe with enhanced stability and sufficient half-life to elucidate the long-term (tumoral) albumin homeostasis. [89Zr]Zr-DFO*malHSA demonstrated excellent metabolic stability and high tumor uptake in a longitudinal PET study (72 h p.i.) using a subcutaneous colorectal cancer allograft model (CT26). Preliminary results also showed enhanced enrichment of the PET probe in an intraperitoneally injected CT26 model indicating the role of the EPR effect not only in subcutaneous models. Consequently, [89Zr]Zr-DFO*malHSA is a promising tool to image albumin accumulation in malignant tissues and should be further (pre)clinically developed as a companion diagnostic agent for patient stratification in trials with albumin-binding drugs.

Platinum(II) complexes prevail as first-line treatment for many cancers but are associated with serious side effects and resistance development. Picoplatin emerged as a promising alternative to circumvent GSH-induced tumor resistance by introducing a bulky 2-picoline ligand. Although clinical studies were encouraging, picoplatin did not receive approval. Interestingly, the anticancer potential of prodrugs based on picoplatin is widely underexplored, and even less so the respective tumor-targeting approaches. We synthesized two new "hybrid" picoplatin(II) derivatives with an oxalate or cyclobutane dicarboxylate leaving group and their corresponding platinum(IV) prodrugs with an albumin-targeting maleimide moiety or a succinimide as reference. Picoplatin(II) and its derivatives indeed reacted much slower with GSH compared to the respective analogs cisplatin, carboplatin, or oxaliplatin. While PicoCarbo(IV) and PicoOxali(IV) were reduced slowly in the presence of ascorbic acid, picoplatin(IV) was extremely unstable. All three prodrugs were widely inactive in the MTT assays. The platinum(IV)-maleimide complexes rapidly bound to albumin with stable conjugates for >25 h. Albumin-binding resulted in elevated platinum plasma levels, prolonged blood circulation, and enhanced tumor accumulation of the prodrugs in mice bearing CT26 tumors. However, only maleimide-functionalized PicoCarbo(IV) and picoplatin(II) significantly inhibited tumor growth. One possible explanation is that for albumin-binding platinum(IV) prodrugs, the bulky 2-picoline moiety prevents sufficient activation/reduction to unlock their full anticancer potential.

The anticancer thiosemicarbazone Triapine is currently in a phase III clinical trial in combination with radiation therapy and cisplatin. Noteworthy, while radiotherapy induces an immune-activating cell death, so called immunogenic cell death (ICD), cisplatin possesses immunomodulatory and ICD-enhancing functions. Interestingly, although there are several indications that suggest that Triapine could also enhance the immune recognition of cancer cells, no investigations in this direction have been reported so far. Indeed, immune cells (especially cytotoxic T-cells) were found to enhance the anticancer activity of Triapine. This effect might be based on endoplasmic reticulum (ER) stress induction, which on the one hand led to ICD of the cancer cells as indicated by ATP release, calreticulin exposure, high-mobility group box 1 secretion and in vivo vaccination experiments. On the other hand, the Triapine-induced ER stress resulted in FAS upregulation in cell culture as well as in vivo via NFκB signaling. This, in turn, rendered cancer cells more susceptible to FASL (predominantly expressed by lymphoid immune cells)-induced caspase 8-mediated apoptosis. Consequently, our study is the first to unveil the significant role of the (adaptive) immune system in the anticancer activity of Triapine, positioning it as a promising partner for combination with immunotherapy and other immunogenic agents.

Maleimides remain very popular conjugation moieties in the fields of bio(in)organic chemistry and biotechnology. They are particularly interesting for endogenous albumin binding in the bloodstream to exploit the enhanced permeability and retention (EPR) effect and to increase tumor accumulation of anticancer drugs. However, during drug development, insufficient aqueous solubility is frequently a limiting factor. In the present study, four new maleimide linkers were synthesized containing a water-soluble piperazine scaffold. Respective maleimide-platinum(IV)-acetato complexes demonstrated similar hydrolytic stability, albumin-binding kinetics, in vivo serum pharmacokinetics and tissue distribution compared to a reference platinum(IV)-PEG4-maleimide complex. To test the aqueous solubility, platinum(IV)-maleimide complexes containing the highly lipophilic drug ibuprofen were synthesized. Indeed, the compounds containing the new piperazine linkers displayed increased solubility (up to 370 mM) in different aqueous media, whereas the PEG4-maleimide reference was only marginally soluble. Finally, the synthetic toolbox of the new piperazine maleimides was also expanded to pure organic derivatives by conjugation to valine-citrulline-para-aminobenzyl-OH derivatives via peptide and thiourea bonds.

Maleimides are widely used in anticancer drug development for linking small-molecule drugs to macromolecules like antibodies or proteins via thiol-Michael addition reactions. Despite their widespread use, even in clinically approved therapeutics, they present significant drawbacks such as hydrolysis at physiological pH and instability of the formed thiosuccinimide bond. Hence, there is a growing need for more stable yet equally efficient binding units. This is particularly important for drug-delivery systems that bind to endogenous albumin in vivo, exploiting the ability of the protein to accumulate in tumor tissue. This study compares phenyloxadiazolyl methyl sulfone (PODS) and a 2,4-difluorophenyl sulfonamide (DFSA) derivative with maleimide as endogenous albumin binders. Of note, PODS and maleimide bind to Cys34, whereas DFSA targets Lys64 of albumin. The albumin binders were conjugated as axial ligands to oxaliplatin-(IV) complexes (PODS-Ox-OAc and DFSA-Ox-OAc) and studied in comparison to a maleimide-bearing reference compound (Mal-Ox-OAc). Both PODS- and DFSA-complexes showed higher hydrolytic stability at pH 7.4 than the maleimide complex. Albumin-binding was highly efficient for the PODS and maleimide complexes. However, the DFSA derivative exhibited only slow conjugation. This was also reflected in the serum pharmacokinetic and organ distribution studies using CT26 colon cancer-bearing mice. Here, the PODS complex showed the highest platinum levels in both serum and tumor tissue. Additionally, PODS-Ox-OAc induced the most significant tumor regression and prolonged overall survival in this model. Together, our data highlight PODS as a promising alternative to maleimide as an endogenous albumin binder.

Several Cu-ligands, including 1,10-phenanthroline (Phen), have been investigated for anticancer purposes based on their capacity to bind excess Cu in cancer tissues and form redox active complexes able to catalyse the formation of reactive oxygen species (ROS), ultimately leading to oxidative stress and cell death. However, the stability and pro-oxidant activity of Cu-based drugs such as Cu-Phen2 is affected in most cell compartments (e.g. cytosol and nucleus) by the presence of compounds such as glutathione (GSH) and metallothioneins, which can reduce and dissociate Cu(II) from the ligand forming poorly redox-active Cu(I)-thiolate clusters. Here, cell culture studies suggested that lysosomal acidification may play a pivotal role in the anticancer activity of Cu-Phen2. In addition, ROS generation catalysed by Cu-Phen2 in the presence of GSH was shown to be remarkably accelerated at the acidic pH typical of lysosomes. The catalytic mechanism was thoroughly investigated by means of density functional theory (DFT) calculations, which disclose key reaction intermediate species, including a ternary Phen-Cu-GSH complex formed upon dissociation of one Phen ligand. Spectroscopic measurements (including low-temperature luminescence, UV-vis absorption and X-ray absorption spectroscopy) corroborated the formation of such a reactive intermediate ternary complex. Furthermore, they revealed that the faster ROS generation observed at lower pH is due to a pH-dependent competition between Phen and GSH for Cu, which results in a higher stability of Cu-Phen2 against dissociation and de-activation by GSH at lower pH. Overall, this study points to lysosomal targeting as an innovative and effective strategy to improve the stability and cytotoxic activity of Cu-based drugs.

Several copper-ligands, including 1,10-phenanthroline (Phen), have been investigated for anticancer purposes based on their capacity to bind excess copper (Cu) in cancer tissues and form redox active complexes able to catalyse the formation of reactive oxygen species (ROS), ultimately leading to oxidative stress and cell death. Glutathione (GSH) is a critical compound as it is highly concentrated intracellularly and can reduce and dissociate copper(II) from the ligand forming poorly redox-active copper(I)-thiolate clusters. Here we report that Cu-Phen 2 speciation evolves in physiologically relevant GSH concentrations. Experimental and computational experiments suggest that at pH 7.4 mostly copper(I)-GSH clusters are formed, but a minor species of copper(I) bound to one Phen and forming ternary complexes with GSH (GS−Cu-Phen) is the redox active species, oxidizing quite efficiently GSH to GSSG and forming HO⋅ radicals. This minor active species becomes more populated at lower pH, such as typical lysosomal pH 5, resulting in faster GSH oxidation and HO⋅ production. Consistently, cell culture studies showed lower toxicity of Cu-Phen 2 upon inhibition of lysosomal acidification. Overall, this study underscores that sub-cellular localisation can considerably influence the speciation of Cu-based drugs and that minor species can be the most redox- and biologically-active.

Abstract Platinum(II)-based chemotherapeutics are among the most commonly used anticancer drugs and are part of nearly every second treatment scheme. However, they lack in tumor specificity, causing severe sides effects, dose-dependent toxicity as well as drug resistance development. Due to their higher tolerability, platinum(IV) prodrugs are currently in the focus of interest. However, comparable to their platinum(II) counterparts, they show insufficient tumor accumulation and are frequently prematurely activated. A promising strategy to improve tumor targeting of anticancer drugs is to exploit the enhanced consumption and accumulation of albumin in the malignant tissue. Thus, we developed a new albumin-targeted maleimide-containing platinum(IV) prodrug, which releases carboplatin in a highly tumor-specific manner. The maleimide moieties enable the drug to selectively bind the endogenous albumin via its free thiol of cysteine 34. The aim of this study was to in-depth characterize the pharmacological behavior and anticancer activity in vivo of the new prodrug. To this end, several xenograft and allograft experiments were performed. To investigate tissue distribution and pharmacokinetics serum, urine, tumor and organ samples of mice were collected after drug treatment and were evaluated by (size-exclusion chromatography) inductively-coupled plasma mass spectrometry and immunohistochemistry (e.g cleaved caspase-3). These experiments revealed that the new prodrug fast and selectively binds to the serum albumin after intravenous injection, leading to an enhanced plasma half-life and an increased tumor accumulation in comparison to its corresponding platinum(II) drug carboplatin. Additionally, the new complex resulted in superior anticancer activity and prolonged overall survival based on the distinctly improved pharmacokinetic profile and enhanced apoptosis induction. In conclusion, these data support that albumin binding is a potent tool to increase the tumor specificity of platinum(IV) drugs and prevent their premature activation in other body compartments. Consequently, this very promising prodrug will be further developed towards clinical phase I testing. Citation Format: Hemma Schueffl, Regina Weinmuellner, Nadine Sommerfeld, Bernhard K. Keppler, Walter Berger, Christian R. Kowol, Petra Heffeter. Improving carboplatin therapy: A novel albumin-targeted platinum(IV) prodrug with superior anticancer activity in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4528.

Au I -carbene and Pt IV −Au I -carbene prodrugs display low to sub-μM activity against several cancer cell lines and overcome cisplatin (cisPt) resistance. Linking a cisPt-derived Pt IV (phenylbutyrate) complex to a Au I -phenylimidazolylidene complex 2 , yielded the most potent prodrug. While in vivo tests against Lewis Lung Carcinoma showed that the prodrug Pt IV (phenylbutyrate)-Au I -carbene ( 7 ) and the 1 : 1 : 1 co-administration of cisPt: phenylbutyrate: 2 efficiently inhibited tumor growth (≈95 %), much better than 2 (75 %) or cisPt (84 %), 7 exhibited only 5 % body weight loss compared to 14 % for 2 , 20 % for cisPt and >30 % for the co-administration. 7 was much more efficient than 2 at inhibiting TrxR activity in the isolated enzyme, in cells and in the tumor, even though it was much less efficient than 2 at binding to selenocysteine peptides modeling the active site of TrxR. Organ distribution and laser-ablation (LA)-ICP-TOFMS imaging suggest that 7 arrives intact at the tumor and is activated there.

Introduction: The prostaglandin E2 (PGE2) pathway is one of the main mediators of intestinal inflammation. As activation of the calcium-sensing receptor (CaSR) induces expression of inflammatory markers in the colon, we assessed the impact of the CaSR on the PGE2 pathway regulation in colon cancer cells and the colon in vitro and in vivo.Methods and Results: We treated CaSR-transfected HT29 and Caco-2 colon cancer cell lines with different orthosteric ligands or modulators of the CaSR and measured gene expression and PGE2 levels. In CaSR-transfected HT29CaSR-GFP and Caco-2CaSR-GFP cells, the orthosteric CaSR ligand spermine and the positive allosteric CaSR modulator NPS R-568 both induced an inflammatory state as measured by IL-8 gene expression and significantly increased the expression of the PGE2 pathway key enzymes cyclooxygenase (COX)-2 and/or prostaglandin E2 synthase 1 (PGES-1). Inhibition of the CaSR with the calcilytic NPS 2143 abolished the spermine- and NPS R-568-induced pro-inflammatory response. Interestingly, we observed cell-line specific responses as e.g. PGES-1 expression was affected only in HT29CaSR-GFP but not in Caco-2CaSR-GFP cells. Other genes involved in the PGE2 pathway (COX-1, or the PGE2 receptors) were not responsive to the treatment. None of the studied genes were affected by any CaSR agonist in GFP-only transfected HT29GFP and Caco-2GFP cells, indicating that the observed gene-inducing effects of spermine and R-568 were indeed mediated by the CaSR.In vivo, we had previously determined that treatment with the clinically approved calcimimetic cinacalcet worsened symptoms in a dextran sulfate sodium (DSS)-induced colitis mouse model. In the colons of these mice, cinacalcet significantly induced gene expression of PGES-2 and the EP3 receptor, but not COX-2; while NPS 2143 increased the expression of the PGE2-degrading enzyme 15-hydroxyprostaglandin dehydrogenase (15-PGDH). Importantly, neither treatment had any effect on the colons of non-DSS treated mice. Discussion: Overall, we show that activation of the CaSR induces the PGE2 pathway, albeit with differing effects in vitro and in vivo. This may be due to the different microenvironment in vivo compared to in vitro, specifically the presence of a CaSR-responsive immune system. Since calcilytics inhibit ligand-mediated CaSR signaling, they may be considered for novel therapies against inflammatory bowel disease.

Small-molecule EGFR inhibitors have distinctly improved the overall survival especially in EGFR-mutated lung cancer. However, their use is often limited by severe adverse effects and rapid resistance development. To overcome these limitations, a hypoxia-activatable Co(III)-based prodrug (KP2334) was recently synthesized releasing the new EGFR inhibitor KP2187 in a highly tumor-specific manner only in hypoxic areas of the tumor. However, the chemical modifications in KP2187 necessary for cobalt chelation could potentially interfere with its EGFR-binding ability. Consequently, in this study, the biological activity and EGFR inhibition potential of KP2187 was compared to clinically approved EGFR inhibitors. In general, the activity as well as EGFR binding (shown in docking studies) was very similar to erlotinib and gefitinib (while other EGFR-inhibitory drugs behaved different) indicating no interference of the chelating moiety with the EGFR binding. Moreover, KP2187 significantly inhibited cancer cell proliferation as well as EGFR pathway activation in vitro and in vivo. Finally, KP2187 proved to be highly synergistic with VEGFR inhibitors such as sunitinib. This indicates that KP2187-releasing hypoxia-activated prodrug systems are promising candidates to overcome the clinically observed enhanced toxicity of EGFR-VEGFR inhibitor combination therapies.

Platinum(IV) prodrugs are highly interesting alternatives to platinum(II) anticancer therapeutics due to their increased tumor selectivity and reduced side effects. In contrast to the established theory, we recently observed that the equatorial ligand(s) of e.g. oxaliplatin(IV) complexes can be hydrolyzed with formation of [(DACH)Pt(OHeq )2 (OAcax )2 ]. In the work presented here, we investigated the reactivity and synthetic usability of this complex to be exploited as a precursor for the development of novel platinum(IV) complexes, not able to be synthesized by conventional protocols. Indeed, we could substitute the equatorial hydroxido ligand(s) e.g. by one or two monodentate biotin ligands (which would be oxidized under standard methods). The formed complexes turned out to be very stable with slow ligand release after reduction, ideal for long-circulating tumor-targeting strategies. Therefore, two platinum(IV) complexes with equatorial maleimides, capable of exploiting serum albumin as a natural nanocarrier, were synthesized as well. The complexes showed massively prolonged plasma half-life and distinctly improved anticancer activity in vivo compared to oxaliplatin. Taken together, the newly developed synthetic platform allows the simple and specific insertion of equatorial ligands into platinum(IV) complexes. This will enable the attachment of three different (bioactive) moieties generating targeted triple-action platinum(IV) prodrugs within one single platinum complex.

AbstractBottlebrush polymers are highly promising as unimolecular nanomedicines due to their unique control over the critical parameters of size, shape and chemical function. However, since they are prepared from biopersistent carbon backbones, most known bottlebrush polymers are non‐degradable and thus unsuitable for systemic therapeutic administration. Herein, we report the design and synthesis of novel poly(organo)phosphazene‐g‐poly(α‐glutamate) (PPz‐g‐PGA) bottlebrush polymers with exceptional control over their structure and molecular dimensions (Dh ≈ 15–50 nm). These single macromolecules show outstanding aqueous solubility, ultra‐high multivalency and biodegradability, making them ideal as nanomedicines. While well‐established in polymer therapeutics, it has hitherto not been possible to prepare defined single macromolecules of PGA in these nanosized dimensions. A direct correlation was observed between the macromolecular dimensions of the bottlebrush polymers and their intracellular uptake in CT26 colon cancer cells. Furthermore, the bottlebrush macromolecular structure visibly enhanced the pharmacokinetics by reducing renal clearance and extending plasma half‐lives. Real‐time analysis of the biodistribution dynamics showed architecture‐driven organ distribution and enhanced tumor accumulation. This work, therefore, introduces a robust, controlled synthesis route to bottlebrush polypeptides, overcoming limitations of current polymer‐based nanomedicines and, in doing so, offers valuable insights into the influence of architecture on the in vivo performance of nanomedicines.

For a variety of cancer types, platinum compounds are still among the best treatment options. However, their application is limited by side effects and drug resistance. Consequently, multi-targeted platinum(IV) prodrugs that target specific traits of the malignant tissue are interesting new candidates. Recently, cisPt(PhB)2 was synthesized which, upon reduction in the malignant tissue, releases phenylbutyrate (PhB), a metabolically active fatty acid analog, in addition to cisplatin. In this study, we in-depth investigated the anticancer properties of this new complex in cell culture and in mouse allograft experiments. CisPt(PhB)2 showed a distinctly improved anticancer activity compared to cisplatin as well as to PhB alone and was able to overcome various frequently occurring drug resistance mechanisms. Furthermore, we observed that differences in the cellular fatty acid metabolism and mitochondrial activity distinctly impacted the drug’s mode of action. Subsequent analyses revealed that “Warburg-like” cells, which are characterized by deficient mitochondrial function and fatty acid catabolism, are less capable of coping with cisPt(PhB)2 leading to rapid induction of a non-apoptotic form of cell death. Summarizing, cisPt(PhB)2 is a new orally applicable platinum(IV) prodrug with promising activity especially against cisplatin-resistant cancer cells with “Warburg-like” properties.

AbstractPlatin(IV)‐Prodrugs sind aufgrund ihrer erhöhten Tumorselektivität und geringeren Nebenwirkungen äußerst interessante Alternativen zu Platin(II)‐Antitumortherapeutika. Im Gegensatz zur gängigen Theorie haben wir kürzlich beobachtet, dass äquatoriale Liganden von z. B. Oxaliplatin(IV)‐Komplexen unter Bildung von [(DACH)Pt(OHeq)2(OAcax)2] hydrolysiert werden können. In der hier vorgestellten Arbeit untersuchten wir die Reaktivität und synthetische Verwendbarkeit dieses Komplexes, als Vorstufe für die Entwicklung neuartiger Platin(IV)‐Komplexe, welche mit herkömmlichen Methoden nicht zugänglich sind. Tatsächlich war es möglich die äquatorialen Hydroxidoliganden z. B. durch ein oder zwei monodentate Biotin‐Liganden, die unter Standardmethoden oxidiert werden würden, zu ersetzen. Die gebildeten Komplexe erwiesen sich als sehr stabil und zeigten auch nach der Reduktion eine langsame Ligandenfreisetzung, eine ideale Eigenschaft für lang zirkulierende zielgerichtete Strategien. Daraufhin wurden zwei Platin(IV)‐Komplexe mit äquatorialen Maleimiden, für die Bindung an Serumalbumin als natürlichen Nanocarrier, synthetisiert. Die Komplexe zeigten im Vergleich zu Oxaliplatin eine stark verlängerte Plasmahalbwertszeit und eine deutlich verbesserte Antitumoraktivität in vivo. Zusammenfassend ermöglicht diese neu entwickelte Syntheseplattform den einfachen und gezielten Einbau äquatorialer Liganden in Platin(IV)‐Komplexe. Des Weiteren können verschiedene (bioaktive) Einheiten koordiniert werden, wodurch sogar zielgerichtete dreifach‐wirksame Platin(IV)‐Prodrugs mit nur einem Platinzentrum möglich wären.

2Malignant tissue is characterized by several very specific traits, which offer the opportunity for the development of drugs with increased tumor specificity and tolerability. Here especially a changed redox homeostasis, altered blood supply, the occurrence of low oxygenated areas (hypoxia), decreased pH values, and specific enzyme expression need to be considered. Since several decades, metal compounds (especially Pt drugs) are key players in the daily therapy of cancer patients. The chemistry of diverse metal drugs has several characteristics, which allow the design of tumor-specific activated prodrugs. Of particular note is the activation by reduction principle, which is based on chemical changes induced by reduction of the central metal ion, representing a very 3elegant and powerful strategy for, e.g., Pt, Ru, Cu, and Co (pro)drugs. This chapter aims to give an overview of metal-based prodrugs and their nanoformulations, which have been designed to be activated by tumor-specific stimuli.

Glutathione (GSH) is the most abundant thiol in mammalian cells and plays a crucial role in maintaining the redox state of many biomolecules, in detoxification and in antioxidant defence. The thiols of two GSH molecules can be oxidized to the disulphide GSSG. The cytosolic GSH/GSSG ratio is very high (> 100), and its decrease can lead to apoptosis or necrosis, which are of interest in cancer research. CuII ions are very efficient oxidants of thiols, but with an excess of GSH, the formed CuIn(GS)m clusters are only slowly re-oxidized by O2 at pH 7.4, and even more slowly at lower pH. Here, the oxidation of GSH by CuII was investigated in the presence of the anticancer thiosemicarbazone Dp44mT. The results showed that CuII-Dp44mT oxidizes GSH faster than CuII alone at pH 7.4, and hence accelerates the production of the very reactive hydroxyl radicals. Interestingly, the GSH oxidation and hydroxyl radical production by CuII-Dp44mT were accelerated at the acidic pH found in lysosomes, where CuII-Dp44mT was previously shown to accumulate and induce lysosomal membrane permeabilization. To decipher this unusually faster thiol oxidation a lower pH, Density Functional Theory (DFT) calculations and spectroscopic studies were performed. The results suggest that the acceleration is due to the protonation of CuII-Dp44mT on the hydrazinic nitrogen, which favours the rate-limiting reduction step without subsequent dissociation of the CuI-complex. Furthermore, preliminary biological studies in cell culture using the proton pump inhibitor bafilomycin A1 indicated that the lysosomal pH plays a role in the activity of Dp44mT.

CD47 protects healthy cells from macrophage attack by binding to signal regulatory protein α (SIRPα), while its upregulation in cancer prevents immune clearance. Systemic treatment with CD47 antibodies requires a weakened Fc-mediated effector function or lower CD47-binding affinity to prevent side effects. Our approach combines "the best of both worlds," i.e., maximized CD47 binding and full Fc-mediated immune activity, by exploiting gene therapy for paracrine release. We developed a plasmid vector encoding for the secreted fusion protein sCV1-hIgG1, comprising highly efficient CD47-blocking moiety CV1 and Fc domain of human immunoglobulin G1 (IgG1) with maximized immune activation. sCV1-hIgG1 exhibited a potent bystander effect, blocking CD47 on all cells via fusion protein secreted from only a fraction of cells or when transferring transfection supernatant to untransfected cells. The CpG-free plasmid ensured sustained secretion of sCV1-hIgG1. In orthotopic human triple-negative breast cancer in CB17-severe combined immunodeficiency (SCID) mice, ex vivo transfection significantly delayed tumor growth and eradicated one-third of tumors. In intratumoral transfection experiments, CD47 blockage and increased migration of macrophages into the tumor were observed within 17 h of a single injection. Natural killer (NK) cell-mediated lysis of sCV1-hIgG1-expressing cells was demonstrated in vitro. Taken together, this approach also opens the opportunity to block, in principle, any immune checkpoints.

Chemotherapy with platinum complexes is essential for clinical anticancer therapy. However, due to side effects and drug resistance, further drug improvement is urgently needed. Herein, we report on triple-action platinum(IV) prodrugs, which, in addition to tumor targeting via maleimide-mediated albumin binding, release the immunomodulatory ligand 1-methyl-d-tryptophan (1-MDT). Unexpectedly, structure-activity relationship analysis showed that the mode of 1-MDT conjugation distinctly impacts the reducibility and thus activation of the prodrugs. This in turn affected ligand release, pharmacokinetic properties, efficiency of immunomodulation, and the anticancer activity in vitro and in a mouse model in vivo. Moreover, we could demonstrate that the design of albumin-targeted multi-modal prodrugs using platinum(IV) is a promising strategy to enhance the cellular uptake of bioactive ligands with low cell permeability (1-MDT) and to improve their selective delivery into the malignant tissue. This will allow tumor-specific anticancer therapy supported by a favorably tuned immune microenvironment.