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BackgroundThe tumor microenvironment (TME) poses challenges that limit the efficacy of conventional CAR-T cell therapies. Homing barriers, immunosuppressive factors, and target antigen heterogeneity can impair CAR-T cell functional activity within the TME. Alternative strategies have contemplated incorporating the use of gamma delta (γδ) T cells as a CAR-T cell approach to potentially overcome these limitations. γδ T cells possess both innate and adaptive immunity to facilitate broad tumor recognition, and their natural propensity for tissue tropism may allow for more effective tumor infiltration. Reported here is the preclinical characterization of ADI-270, an allogeneic γδ CAR-T cell product targeting CD70+ cancers, engineered with a third-generation CAR based on the natural CD27 receptor. ADI-270 is also double-armored to mitigate the immunosuppressive effects of TGFβ and reduce the potential for allogeneic rejection.MethodsVδ1 T cells engineered to express an anti-CD70 CAR and dominant negative TGFβ receptor II (dnTGFβRII) were expanded from healthy donor human PBMCs. The phenotype and functional characterization of ADI-270 were assessed with in vitro cell culture assays and in vivo tumor xenograft models.ResultsADI-270 exhibited high levels of in vitro cytotoxicity against a panel of cancer cell lines and displayed a favorable inflammatory cytokine profile compared with reference scFv-based anti-CD70 CAR αβ T cells. Cytotoxicity remained potent despite low CD70 expression observed in multiple solid and hematologic tumor cell models. When armored with dnTGFβRII, ADI-270 exhibited functional resilience to TGFβ-mediated inhibition of T cell effector activity. In addition, the incorporation of potent and sensitive CD70-targeting decreased T cell-mediated alloreactive killing against ADI-270 in vitro without evidence of fratricide. Finally, ADI-270 displayed robust tumor tropism and control of primary and secondary tumor challenges in xenograft mouse models.ConclusionsThese results demonstrate the robust potency and capacity of ADI-270 to extend antitumor activity to cancers with heterogeneous antigen expression. The functional armoring incorporated into ADI-270 provides a mechanism to overcome the limitations of reduced efficacy and persistence within the TME. ADI-270 has the potential to target multiple CD70+ cancers with initial clinical evaluation proceeding in relapsed/refractory clear cell renal cell carcinoma.Trial registration numberNCT06480565.

Long-acting nanoformulated antiretroviral therapy (nanoART) induces a range of innate immune migratory, phagocytic and secretory cell functions that perpetuate drug depots. While recycling endosomes serve as the macrophage subcellular depots, little is known of the dynamics of nanoART-cell interactions. To this end, we assessed temporal leukocyte responses, drug uptake and distribution following both intraperitoneal and intramuscular injection of nanoformulated atazanavir (nanoATV). Local inflammatory responses heralded drug distribution to peritoneal cell populations, regional lymph nodes, spleen and liver. This proceeded for three days in male Balb/c mice. NanoATV-induced changes in myeloid populations were assessed by fluorescence-activated cell sorting (FACS) with CD45, CD3, CD11b, F4/80, and GR-1 antibodies. The localization of nanoATV within leukocyte cell subsets was determined by confocal microscopy. Combined FACS and ultra-performance liquid chromatography tandem mass-spectrometry assays determined nanoATV carriages by cell-based vehicles. A robust granulocyte, but not peritoneal macrophage nanoATV response paralleled zymosan A treatment. ATV levels were highest at sites of injection in peritoneal or muscle macrophages, dependent on the injection site. The spleen and liver served as nanoATV tissue depots while drug levels in lymph nodes were higher than those recorded in plasma. Dual polymer and cell labeling demonstrated a nearly exclusive drug reservoir in macrophages within the liver and spleen. Overall, nanoART induces innate immune responses coincident with rapid tissue macrophage distribution. Taken together, these works provide avenues for therapeutic development designed towards chemical eradication of human immunodeficiency viral infection.

While the therapeutic potential for current long-acting (LA) antiretroviral therapy (ART) is undeniable, ligand-decorated nanoformulated LA-ART could optimize drug delivery to viral reservoirs. The development of decorated ART hinges, however, on formulation processes and manufacture efficiencies. To this end, we compared manufacture and purification techniques for ligand-decorated antiretroviral drug nanocrystals. Ligand-decorated nanoparticle manufacturing was tested using folic acid (FA) nanoformulated cabotegravir. Direct manufacturing of FA-cabotegravir resulted in stable particles with high drug loading and monocyte-macrophage targeting. A one step 'direct' scheme proved superior over differential centrifugation or tangential flow filtration facilitating particle stability and preparation simplicity and efficiency. Direct manufacturing of FA nanoparticles provides a path toward large-scale clinical grade manufacturing of cell-targeted LA-ART. Folic acid (FA) decoration on the surface of nanocrystals can be achieved by mixing FA conjugated poloxamer 407 (FA-P407) and native P407 in varied ratios followed by size reduction by homogenization and differential centrifugation or tangential flow filtration to remove excess unbound polymers. The optimized manufacturing scheme is by direct homogenization with predetermined quantity of FA conjugated P407. Direct manufacturing method yields stable homogenous nanoparticles with high drug loading.

Regimen adherence, systemic toxicities, and limited drug penetrance to viral reservoirs are obstacles limiting the effectiveness of antiretroviral therapy (ART). Our laboratory's development of the monocyte-macrophage-targeted long-acting nanoformulated ART (nanoART) carriage provides a novel opportunity to simplify drug-dosing regimens. Progress has nonetheless been slowed by cumbersome, but required, pharmacokinetic (PK), pharmacodynamics, and biodistribution testing. To this end, we developed a small magnetite ART (SMART) nanoparticle platform to assess antiretroviral drug tissue biodistribution and PK using magnetic resonance imaging (MRI) scans. Herein, we have taken this technique a significant step further by determining nanoART PK with folic acid (FA) decorated magnetite (ultrasmall superparamagnetic iron oxide [USPIO]) particles and by using SMART particles. FA nanoparticles enhanced the entry and particle retention to the reticuloendothelial system over nondecorated polymers after systemic administration into mice. These data were seen by MRI testing and validated by comparison with SMART particles and direct evaluation of tissue drug levels after nanoART. The development of alendronate (ALN)-coated magnetite thus serves as a rapid initial screen for the ability of targeting ligands to enhance nanoparticle-antiretroviral drug biodistribution, underscoring the value of decorated magnetite particles as a theranostic tool for improved drug delivery.

Previous efforts from our laboratory were successful in harnessing mononuclear phagocytes (MP; monocytes, dendritic cells and macrophages) for nanoformulated drug transport, delivery and distribution. This is particularly noteworthy for nanoformulated antiretroviral therapy (nanoART) as the paths traveled by MP parallel the virus itself. Thus, using MP as Trojan horses for drug delivery could facilitate virus elimination in its target tissues. Prior works showed that ritonavir (RTV), atazanavir (ATV) and efavirenz (EFV) nanoparticles facilitated entry, release and antiretroviral activities into and from infected cells. However, the major share of the administered drug was metabolized within 24 hours after parenteral administration. Thus, improving bioavailability and the therapeutic index of the drug formulations is urgently needed. We believe this can be achieved through cell-targeted nanoART that would speed MP uptake and achieve sustained cell and tissue drug depots. To this end, we posit that nanoART coated with folic acid (FA), N-formyl methionine leucine phenylalanine (fMLP) or a mannose-decorated monomer polymer can engage MP receptors to enhance particle entry in subcellular MP compartments during both steady state conditions and following immune activation. Laboratory results were tested in normal (Balb/cJ), Hu-PBL reconstituted NOD/scid-gcnull (NSG) and CD34+ hematopoietic stem cell transplanted humanized mice. The drug biodistribution, pharmacokinetics (PK) and bioavailability were determined in formulation optimization. Improvements in cell uptake, intracellular compartmentalization, release and antiretroviral responses correlated to the physicochemical particle characteristics, ligand coating, biologic properties and cell receptor expression. We believe that finding an appropriate targeting ligand of the newly developed nanoformulations will facilitate clinical translation of antiretroviral nanoformulations.

Drug toxicities, compliance and penetrance into viral reservoirs have diminished the efficacy of long-term antiretroviral therapy (ART) for treatment of HIV infection. Cell-targeted nanoformulated ART was developed to improve disease outcomes. However, rapid noninvasive determination of drug biodistribution is unrealized. To this end, small magnetite ART (SMART) nanoparticles can provide assessments of ART biodistribution by MRI. Poly(lactic- -glycolic acid), 1,2-distearoyl- -glycero-3-phosphocholine- and 1,2-distearoyl- -glycero-3-phosphoethanolamine- -(methoxy-PEG 2000)-encased particles were synthesized with atazanavir (ATV) and magnetite. Uptake and retention of ATV and magnetite administered at 3:1 ratios (weight/weight) were determined in human monocyte-derived macrophages and mice. SMART particles were taken up and retained in macrophages. In mice, following parenteral SMART injection, magnetite and drug biodistribution paralleled one another with MRI signal intensity greatest in the liver and spleen at 24 h. Significantly, ATV and magnetite levels correlated. SMART can permit rapid assessment of drug tissue concentrations in viral reservoirs. Original submitted 11 February 2013; Revised submitted 2 April 2013

Long-acting nanoformulated antiretroviral therapy (nanoART) induces a range of innate immune migratory, phagocytic and secretory cell functions that perpetuate drug depots. While recycling endosomes serve as the macrophage subcellular depots, little is known of the dynamics of nanoART-cell interactions. To this end, we assessed temporal leukocyte responses, drug uptake and distribution following both intraperitoneal and intramuscular injection of nanoformulated atazanavir (nanoATV). Local inflammatory responses heralded drug distribution to peritoneal cell populations, regional lymph nodes, spleen and liver. This proceeded for three days in male Balb/c mice. NanoATV-induced changes in myeloid populations were assessed by fluorescence-activated cell sorting (FACS) with CD45, CD3, CD11b, F4/80, and GR-1 antibodies. The localization of nanoATV within leukocyte cell subsets was determined by confocal microscopy. Combined FACS and ultra-performance liquid chromatography tandem mass-spectrometry assays determined nanoATV carriages by cell-based vehicles. A robust granulocyte, but not peritoneal macrophage nanoATV response paralleled zymosan A treatment. ATV levels were highest at sites of injection in peritoneal or muscle macrophages, dependent on the injection site. The spleen and liver served as nanoATV tissue depots while drug levels in lymph nodes were higher than those recorded in plasma. Dual polymer and cell labeling demonstrated a nearly exclusive drug reservoir in macrophages within the liver and spleen. Overall, nanoART induces innate immune responses coincident with rapid tissue macrophage distribution. Taken together, these works provide avenues for therapeutic development designed towards chemical eradication of human immunodeficiency viral infection.