Engineering Tolerance toward Allogeneic CAR-T Cells by Regulation of MHC Surface Expression with Human Herpes Virus-8 Proteins.

Allogeneic, off-the-shelf (OTS) chimeric antigen receptor (CAR) cell therapies have the potential to reduce manufacturing costs and variability while providing broader accessibility to cancer patients and those with other diseases. However, host-versus-graft reactivity can limit the durability and efficacy of OTS cell therapies requiring new strategies to evade adaptive and innate-immune responses. Human herpes virus-8 (HHV8) maintains infection, in part, by evading host T and natural killer (NK) cell attack. The viral K3 gene encodes a membrane-tethered E3 ubiquitin ligase that discretely targets major histocompatibility complex (MHC) class I components, whereas K5 encodes a similar E3 ligase with broader specificity, including MHC-II and the MHC-like MHC class I polypeptide-related sequence A (MIC-A)- and sequence B (MIC-B)-activating ligands of NK cells. We created γ-retroviruses encoding K3 and/or K5 transgenes that efficiently transduce primary human T cells. Expression of K3 or K5 resulted in dramatic downregulation of MHC-IA (human leukocyte antigen [HLA]-A, -B, and -C) and MHC class II (HLA-DR) cell-surface expression. K3 expression was sufficient for T cells to resist exogenously loaded peptide-MHC-specific cytotoxicity, as well as recognition in one-way allogeneic mixed lymphocyte reactions. Further, in immunodeficient mice engrafted with allogeneic T cells, K3-transduced T cells selectively expanded in vivo. Ectopic K5 expression in MHC class I-, MIC-A+/B+ K562 cells also reduced targeting by primary NK cells. Coexpression of K3 in prostate stem cell antigen (PSCA)-directed, inducible MyD88/CD40 (iMC)-enhanced CAR-T cells did not impact cytotoxicity, T cell growth, or cytokine production against HPAC pancreatic tumor target cells, whereas K5-expressing cells showed a modest reduction in interleukin (IL)-2 production without effect on cytotoxicity. Together, these results support application of these E3 ligases to advance development of OTS CAR-T cell products.

Two-Dimensional Regulation of CAR-T Cell Therapy with Orthogonal Switches.

Use of chimeric antigen receptors (CARs) as the basis of targeted adoptive T cell therapies has enabled dramatic efficacy against multiple hematopoietic malignancies, but potency against bulky and solid tumors has lagged, potentially due to insufficient CAR-T cell expansion and persistence. To improve CAR-T cell efficacy, we utilized a potent activation switch based on rimiducid-inducible MyD88 and CD40 (iMC)-signaling elements. To offset potential toxicity risks by this enhanced CAR, an orthogonally regulated, rapamycin-induced, caspase-9-based safety switch (iRC9) was developed to allow in vivo elimination of CAR-T cells. iMC costimulation induced by systemic rimiducid administration enhanced CAR-T cell proliferation, cytokine secretion, and antitumor efficacy in both in vitro assays and xenograft tumor models. Conversely, rapamycin-mediated iRC9 dimerization rapidly induced apoptosis in a dose-dependent fashion as an approach to mitigate therapy-related toxicity. This novel, regulatable dual-switch system may promote greater CAR-T cell expansion and prolonged persistence in a drug-dependent manner while providing a safety switch to mitigate toxicity concerns.

Evaluation of the toxicity of intravenous delivery of auroshell particles (gold-silica nanoshells).

Gold nanoshells (155 nm in diameter with a coating of polyethylene glycol 5000) were evaluated for preclinical biocompatibility, toxicity, and biodistribution as part of a program to develop an injectable device for use in the photothermal ablation of tumors. The evaluation started with a complete good laboratory practice (GLP) compliant International Organization for Standardization (ISO)-10993 biocompatibility program, including cytotoxicity, pyrogenicity (US Pharmacopeia [USP] method in the rabbit), genotoxicity (bacterial mutagenicity, chromosomal aberration assay in Chinese hamster ovary cells, and in vivo mouse micronucleus), in vitro hemolysis, intracutaneous reactivity in the rabbit, sensitization (in the guinea pig maximization assay), and USP/ISO acute systemic toxicity in the mouse. There was no indication of toxicity in any of the studies. Subsequently, nanoshells were evaluated in vivo by intravenous (iv) infusion using a trehalose/water solution in a series of studies in mice, Sprague-Dawley rats, and Beagle dogs to assess toxicity for time durations of up to 404 days. Over the course of 14 GLP studies, the gold nanoshells were well tolerated and, when injected iv, no toxicities or bioincompatibilities were identified.

Selective nanoparticle-directed ablation of the canine prostate.

BACKGROUND AND OBJECTIVES: Prostate cancer is the most frequent cancer type and the second most common cause of cancer death among US men. This study, adapted a previously reported nanoparticle-directed photothermal treatment of brain tumors to the treatment of prostate disease by using normal canine prostate in vivo, directly injected with a suspension of nanoparticles as a proxy for prostate tumor, and by developing laser dosimetry for prostate which is marginally ablative in native tissue, yet producing photothermal coagulation in prostate tissue containing nanoparticles. METHODS: Canine prostates were exposed by surgical laparotomy and directly injected with suspensions of nanoparticles (nanoshells) and irradiated by a NIR laser source delivered percutaneously by an optical fiber catheter and isotropic diffuser. The photothermal lesions were permitted to resolve for up to 8 days, at which time each animal was euthanized, necropsied, and the prostate taken for histopathological and elemental analysis. RESULTS: Nanoparticles were retained for up to 4 hours in prostate and served as a proxy for prostate tumor. A marginally ablative laser dose of 3.0 W for 3 minutes was developed which would yield 4 mm-radius coagulo-necrotic lesions if nanoparticles were present. CONCLUSION: We have shown that the addition of nanoshells to native tissue, combined with a marginally ablative laser dose can generate ablative thermal lesions, and that the radial extent of the thermal lesions is strictly confined to within ∼4 mm of the optical fiber with sub-millimeter uncertainty. This, in turn, suggests a means of precise tumor ablation with an ability to obviate damage to critical structures limited primarily by the precision with which the optical fiber applicator can be placed. In so doing, it should be possible to realize a precise, nerve bundle and urethra sparing prostate cancer treatment using a minimally invasive, percutaneous approach.

Near-infrared narrow-band imaging of gold/silica nanoshells in tumors.

Gold nanoshells (GNS) are a new class of nanoparticles that can be optically tuned to scatter or absorb light from the near-ultraviolet to near-infrared (NIR) region by varying the core (dielectric silica)/shell (gold) ratio. In addition to spectral tunability, GNS are inert and bioconjugatable, making them potential labels for in vivo imaging and therapy of tumors. We report the use of GNS as exogenous contrast agents for enhanced visualization of tumors using narrow-band imaging (NBI). NBI takes advantage of the strong NIR absorption of GNS to distinguish between blood and nanoshells in the tumor by imaging in narrow wavelength bands in the visible and NIR, respectively. Using tissue-simulating phantoms, we determined the optimum wavelengths to enhance contrast between blood and GNS. We then used the optimum wavelengths for ex vivo imaging of tumors extracted from human colon cancer xenograft bearing mice injected with GNS. Systemically delivered GNS accumulated passively in tumor xenografts by the enhanced permeability and retention (EPR) effect. Ex vivo NBI of tumor xenografts demonstrated heterogeneous distribution of GNS with a clear distinction from the tumor vasculature. The results of this study demonstrate the feasibility of using GNS as contrast agents to visualize tumors using NBI.

In-vivo photoacoustic microscopy of nanoshell extravasation from solid tumor vasculature.

In this study, high resolution backward-mode photoacoustic microscopy (PAM) is used to noninvasively image progressive extravasation and accumulation of nanoshells within a solid tumor in vivo. PAM takes advantage of the strong near-infrared absorption of nanoshells and their extravasation tendency from leaky tumor vasculatures for imaging. Subcutaneous tumors are grown on immunocompetent BALB/c mice. Polyethylene glycol (PEGylated) nanoshells with a peak optical absorption at approximately 800 nm are intravenously administered. With an 800-nm laser source, a prescan prior to nanoshell injection is taken to determine the background that is free of nanoshell accumulation. After injection, the 3-D nanoshell distribution at the tumor foci is monitored by PAM for 6 h. Experimental results show that accumulated nanoshells delineate the tumor position. Nanoshell accumulation is heterogeneous in tumors: more concentrated within the tumor cortex and largely absent from the tumor core. Because nanoshells have been recently demonstrated to enhance thermal therapy of subcutaneous tumors, we anticipate that PAM will be an important aid before, during, and after nanoshell thermal therapy.

Quantitative estimation of gold nanoshell concentrations in whole blood using dynamic light scattering.

We demonstrate a new nondestructive optical assay to estimate submicron solid particle concentrations in whole blood. We use dynamic light scattering (DLS), commonly used to estimate nanoparticle characteristics such as size, surface charge, and degree of aggregation, to quantitatively estimate concentration and thereby estimate the actual delivered dose of intravenously injected nanoparticles and the longitudinal clearance rate. Triton X-100 is added to blood samples containing gold (Au) nanoshells to act as a quantitative scattering standard and blood lysing agent. The concentration of nanoshells was determined to be linearly proportional (R(2) = 0.998) to the relative light scattering attributed to nanoshells via DLS as compared with the Triton X-100 micelles in calibration samples. This relationship was found to remain valid (R(2) = 0.9) when estimating the concentration of circulating nanoshells in 15-muL blood samples taken from a murine tumor model as confirmed by neutron activation analysis. Au nanoshells are similar in size and shape to other types of nanoparticles delivered intravascularly in biomedical applications, and given the pervasiveness of DLS in nanoscale particle manufacturing, this simple technique should have wide applicability toward estimating the circulation time of other solid nanoparticles.

In Vivo Detection of Gold Nanoshells in Tumors Using Diffuse Optical Spectroscopy.

This study demonstrates the use of diffuse optical spectroscopy (DOS) for the noninvasive measurement of gold nanoshell concentrations in tumors of live mice. We measured the diffuse optical spectra (500-800 nm) using an optical fiber probe placed in contact with the tissue surface. We performed in vitro studies on tissue phantoms illustrating an accurate measurement of gold-silica nanoshell concentration within 12.6% of the known concentration. In vivo studies were performed on a mouse xenograft tumor model. DOS spectra were measured at preinjection, immediately postinjection, 1 and 24 h postinjection times, and the nanoshell concentrations were verified using neutron activation analysis.