September 1, 2010 (Vol. 30, No. 15)

T-Cell Based Delivery Systems and New Imaging Options Push Field Forward

Nanotechnology is combining with T-cell biology to produce diagnostics and therapeutics that evade the immune system and deliver localized therapeutic payloads, which, through their tight targeting, minimize adverse side effects.

At TechConnect World’s recent “Nanotech” conference and expo in Anaheim, industry and academic researchers elaborated on the advances that are driving nanotechnology toward  the clinic, including T-cell based delivery systems, the ability to design nanoparticles’ function and morphology, and new imaging options.

InCellerate, a new company formed by Laurence J. N. Cooper, M.D., Ph.D., director of pediatric cell therapy at M.D. Anderson Cancer Center, has been working on a high-throughput microelectroporation device—called HiTMeD—to electro-transfer RNA species to redirect T-cell specificity.

The device is also being designed to electro-transfer gold nanoparticles that can be functionalized to enhance T-cell biology. This combination generates T cells that can respond more rapidly and more robustly as a drug-delivery vehicle, thus having potential as a tumor-specific, adoptive immunotherapy.

“We see T cells as a vehicle for targeted delivery of nanoparticles to tissue sites,” Dr. Cooper said. Typically, the reticular endothelial system filters out infused nanoparticles. Wrapping the nanoparticles in T cells thwarts that. And, because chimeric antigen receptor (CAR) expression from mRNA will be temporary, InCellerate developed HiTMeD to make multiple infusions of these modified T cells practical.

T cells endogenously express a panel of receptors that orchestrate their ability to home to disease. InCellerate’s microelectroporation device essentially pokes holes in T cells so genes and/or nanoparticles can be inserted, thus turning the T cells into therapeutic or diagnostic vectors.

“This is different from therapy in which therapeutic genes are introduced using viruses,” Dr. Cooper stressed. The introduced gene enhances specificity to better eliminate malignant cells, while the nanoparticles add potency or imaging properties.

In targeting lung cancer, Dr. Cooper and his colleagues have introduced a CAR into  T cells. This antigen receptor recognizes the CD19 molecule expressed on the cell surface of B-cell malignancies. Introducing the CAR as mRNA avoids integration into the T-cell chromosome and “avoids the potential genotoxicity associated with vector and transgene integration. In addition, the high-throughput capacity overcomes the expected transient CAR expression, as repeated rounds of electroporation can replace T cells that have lost transgene expression.”

HiTMeD can electroporate 2×108 cells within 10 minutes, ex vivo, according to Dr. Cooper. In tests, up to 80% of the primary T cells expressed the CD-19-specific CAR. This is at least a 10-fold improvement over other technologies, he added.

Scientists at InCellerate are working on a high-throughput microelectroporation device called HiTMeD. T cells generated from the device reportedly can respond more rapidly and more robustly as drug-delivery vehicles and also have potential as a tumor-specific, adoptive immunotherapy.

Delivery of Cytotoxic Drugs

At the University of Texas Health Sciences Center at Houston, Ennio Tasciotti, Ph.D., assistant professor in the department of nanomedicine and biomedical engineering and the graduate school for biomedical sciences, has developed a “bio-inspired nanotechnology platform” to deliver cytotoxic drugs or imaging agents to cancer sites more effectively than existing platforms and with fewer side effects.

Dr. Tasciotti explained that by using a multistage silicon nanoparticle delivery system injected subcutaneously, 90% of the drug payload is delivered directly to the target, with only 10% remaining in the bloodstream after six hours.

Traditional delivery methods, in contrast, deliver approximately 10% of the payload to the tissue, thereby contributing to adverse side effects. Within 24 hours, the body’s natural trophism causes 100% of the injected dose of multistage silicon particles to accumulate at the tumor site.

Recent work by Dr. Tasciotti has focused on tight targeting using adipose stromal stem cells derived from human white adipose tissue. By using a patient’s own adipose stem cells, “there’s no rejection and they can be harvested easily with liposuction,” he reported.

“This controlled-release system functions as a Trojan horse, decoupling the homing and therapeutic responsibilities onto separate components.” Basically, diagnostic or therapeutic nanovectors are loaded into the pores of silicon particles, which are decorated with stem cells. Because stem cells respond to the site of inflammation to trigger healing, they are used as targeting agents, Dr. Tasciotti added.

“Stem cells find the tumor site very effectively and accumulate at the tumor.” The silicon shields the nanoparticles from the reticular endothelium system, thus minimizing side effects. 

The technology has been used to target Kaposi’s syndrome, but it is still in its early stages of development. The next step, Dr. Tasciotti said, is to prove good accumulation of the nanoparticles and that they release the drugs at the right time and place before moving to preclinical trials.

Nanoparticle Challenges

One of the challenges in designing nanomaterials that are efficacious for clinical use is the nanoparticles themselves. Historically, it has been difficult to design nanoparticles in which multiple substances are distributed uniformly throughout the particle and then to produce those nanoparticles with a consistent size and shape.

Singapore-based NanoMaterials Technology has overcome that with high gravity controlled precipitation (HGCP). “We can control the size, the shape, and even the crystallinity,” claimed Jimmy Yun, Ph.D., CEO. “We are talking about particle design,” not merely manufacturing nanoparticles. This method ensures the uniform mixing of two solutions, so nucleation can be controlled.

NanoMaterials’ particles are of uniform quality, size, distribution, particle shape, and morphology. Therefore, Dr. Yun explained, their contents behave more predictably than when carried by particles in which the solutions mixed unevenly, which is inherent in manual mixing methods.

In comparing dissolution rates of gravity-controlled precipitation particles with those of spray-dried active ingredients, Dr. Yun said that 80% of the HGCP particles dissolved within 10 minutes, compared to only 20% of the spray-dried particles.

Aerosol performance tests comparing NanoMaterials’ spherical nanoparticles to microsized APIs showed that the total fine-particle fraction for the nanoparticles was nearly 85%, compared to 35% for the microparticles. Additional applications include using these nanoparticles as a controlled-release technology for therapeutics and to induce hyperthermia for tumors (which do not dissipate heat as readily as normal cells).

The company is working at the industrial scale, designing particles as small as 10 nm. The first pharmaceutical pilot plant using HGCP technology can produce 40 tons of antibiotics per year, according to Dr. Yun, and the first commercial production facility can produce 10,000 tons per year.

NanoMaterials just signed a deal to develop particles for a product destined for the FDA-approval process and also has signed a license agreement with a Chinese pharmaceutical firm to develop the particles for State Food and Drug Administration approval.

NanoMaterials Technology’s High Gravity Controlled Precipitation (HGCP) platform facilitates the development of a wide range of nano-sized materials. According to the company, HGCP provides better control over quality, particle size and distribution, particle shape, and morphology of the nanomaterials than spray drying.

Protein Catalytic Capture

James R. Heath, Ph.D., professor of chemistry at California Institute of Technology, is working to advance the understanding of the tumor microenvironment of glioblastoma multiforme cancers.

“Many of the hallmarks of cancer, such as angiogenesis and metastasis, are thought to arise out of a process in which the immune system actually reinforces the aberrant nature of cancer.” As part of that overarching goal, Dr. Heath’s lab is developing a protein measurement instrument that operates at the single-cell level to help unravel the relationship between tumors and the immune system.

“We’re on the cusp of a $1,000 genome, and mRNAs have gotten significantly cheaper, but protein measurement hasn’t,” Dr. Heath said. “We’re trying to develop technology and approaches to do many measurements” simultaneously, at the same cost as a single measurement today.

The goal is to assay a patient’s blood at least daily against a panel of 30 to 50 proteins. Yet, “you can’t ask for blood every few hours,” and lab results typically come back one or two days later, limiting their usefulness in a real-time therapeutic environment. The technology, therefore, must be simple, automated, and require “only a pinprick of blood,” Dr. Heath said. Ideally, results would emerge within one hour.

The miniaturization process is completed. Now Dr. Heath and team are working with immunologists to overcome the limitations of existing assays. The challenge is that the antibodies used in assays all have slight variations among batches, which require instruments to be recalibrated with every change, which requires new proteins that must also be validated. This creates what amounts to a recalibration loop.

To break free of that loop, Dr. Heath is developing assays based upon protein catalyzed capturations, which he described as creating a catalytic scaffold. Essentially, he is creating a peptide library, identifying the binding loads. “Then we take a second group and modify it to have a complementary group.” The result is a catalytic scaffold that catalyzes the covalent coupling between the two groups, an approach that Dr. Heath insists is “very effective.”

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