Yevgeny Brudno

The Aim 1 focus is to develop a viral vector-loaded macroporous scaffold capable of generating CAR-T cells in situ. A biomaterial-based macroporous scaffold will attract and maintain host T cells to a specific microenvironment, where T cells can be transduced with CAR encoded viral vectors, thus generating tumor specific CAR-T cells in situ. The scaffold will then release these CAR-T cells into systemic circulation, where they will destroy target tumors. If successful, this approach could have a huge clinical impact by significantly reduced therapy costs, dramatically expanding the patient population that could benefit from CAR-T-cell therapy. The Aim 2 focus is on refilling drug depots through the combination of non-toxic therapeutic prodrugs. The use of nontoxic prodrug refills allows us to administer large doses to maximally exploit the short window for drug delivery, allowing weeks and potentially months worth of therapeutic to be given. The depots will release active drugs for a long period of time before being non-invasively refilled again. We further propose testing this innovative drug-delivery strategy in a patient-derived tumor model.

Despite unprecedented clinical success of chimeric antigen receptor (CAR)-T cell therapy against tumors, its widespread application is limited by lengthy and labor-intensive ex vivo manufacturing procedures that result in: (i) high cost; (ii) delays to infuse CAR-T cells to patients with rapidly progressing disease; and (iii) heterogeneous composition and terminal differentiation of the infused CAR-T cells that limit their engraftment and persistence. We propose an alternative: The research outlined in this proposal develops a new paradigm to expedite CAR T cell production, enhance CAR T-cell efficacy and, finally, to generate CAR T cells in situ. First, we hypothesize that biomaterial scaffolds displaying anti-CD3/CD28 antibodies and releasing pro-proliferative interleukins will mediate simultaneous activation and viral transduction of T cells under static condition, eliminating expensive and time-consuming activation and transduction protocols and facilitating ex vivo genetic reprogramming of T cells. Second, we hypothesize that when implanted immediately following seeding with PBMCs and CAR encoded viral vectors, we hypothesize that our biomaterial scaffolds will efficiently reprogram and release CAR-T cells into circulation, eliminating ex vivo CAR-T isolation and proliferation protocols and promoting a less differentiated CAR-T cells phenotype and improved CAR-T cell persistence in vivo. Finally, we hypothesize that when encapsulating T cell-attracting cytokines, implanted biomaterial scaffolds will generate CAR T cells entirely in situ through recruitment of host T cells, reprogramming of recruited T cells with resident CAR encoding viral vectors and release of reprogrammed CAR-T cells. We expect our results will provide a basis for a general cellular therapeutic strategy against many types of diseases requiring cellular reprogramming. In addition to the obvious applications in cancer, we expect that our rational materials-based approach for cellular manufacturing to will overcome the rate limiting and labor-intensive manufacturing process of CAR T cell therapy and promote widespread patient access to this therapy, allowing straight-forward adoption to program lymphocytes for other diseases, including viral antigens and disease-associated myocardiocytes.

CAR-T cell therapy has revolutionized the treatment of liquid tumors, including leukemia and lymphoma, and hold enormous promise for treatment of solid cancers as well. However, despite their unprecedented clinical success, widespread utilization of this therapy is hampered by the lengthy and labor-intensive manufacturing procedures. CAR-T cell manufacturing takes weeks, results in very high costs of therapy (~$500,000). The long manufactur-ing time creates delays of weeks or months to infuse CAR-T cells to patients with rapidly progressing disease. Finally, the extensive ex vivo manipulation creates cell products with heterogeneous composition and terminal differentiation that limit CAR-T cell engraftment and persistence. Effort to overcome these limitations have fo-cused on closed and automatic manufacturing devices to contain the labor needed to manufacture CAR-T cells ex vivo, and allogeneic off-the-shelf CAR-T cells have been proposed to overcome the need of CAR-T cell manu-facturing for each single patient. These technologies are promising, but reducing the time, costs and regulatory burden of manufacturing or eliminating ex vivo procedures entirely remains a critical unmet need. In vivo genera-tion of CAR-T cells would eliminate the need for ex vivo procedures, prevent the terminal differentiation of ex vivo expanded CAR-T cells and ensure the potency and longevity of autologous T cells as compared to allogeneic CAR-T cell products that are extensively manipulated to prevent rejection and graft-versus-host disease. This proposal outlines the first steps in a highly innovative high-risk/high-reward effort to develop bioinstructive bio-materials scaffolds that generate CAR-T cells entirely within the patient and produce CAR-T cells with improved efficacy and persistence. Our endeavor is built on significant published and preliminary data demonstrating that our biomaterial scaffolds already efficiently activate and mediate CAR-T cell transduction in vitro and efficiently recruit and release CAR-T cells in vivo and reduce CAR-T manufacturing times from weeks to a single day. We propose that the biocompatible alginate biomaterial scaffolds can be modified to encapsulate T cell-attracting chemokines to recruit T cells to the scaffold. After recruitment, the biomaterial scaffolds will provide ��CD3/CD28 signaling to activate the T cells. After activation, T cell-specific viral particles either already present in the bio-material or administered to the biomaterial as a separate step will transduce the T cells, generating tumor specif-ic CAR-T cells in situ in manner compatible with irradiative lymphodepletion. Finally, interleukin signaling in the scaffold will expand and promote release of formed CAR-T cells for systemic function. If successful, this ap-proach could have enormous clinical impact by significantly reducing therapy costs and dramatically expanding the patient population benefiting from CAR-T-cell therapy. We expect that these studies will provide a founda-tional technology for CAR-T cells manufacturing and promote widespread patient access. In addition to the clear application in cancer, however, this rational, materials-based approach for cellular manufacturing could be adopted to program therapeutic lymphocytes in solid tumors and for other diseases.

Genetic reprogramming of cells could provide cures in a large number of different diseases, include repro-gramming T cells with TCR or CAR to target and kill cancer cells, reprogramming hematopoetic stem cells for mak-ing universal blood products, and reprogramming neural stem cells to improve nerve regeneration. Unfortunately, genetic reprogramming of cells is both labor intensive (~weeks) and expensive (~$100,000). Given the financial bur-den cancer already poses to the healthcare system, providing this precision health therapeutic to millions of cancer patients presents an insurmountable challenge and new manufacturing platforms are desperately needed.

Immunotherapy of cancer with non-thermal plasmas (NTP) is gaining attention for cancer treatment due to its demonstrated therapeutic efficacy in preclinical animal models and its ability to stimulate a robust immune response3������������������6. Although the full mechanism of NTP������������������s anti-cancer effect is still under investigation, it is clear that the dose-dependent effect of NTP treatment is precise, controllable and related to the variety of NTP-generated reactive oxygen and nitrogen species (RONS). The oxidative stress provided by NTP treatment is in some ways similar to radiotherapy, but NTP provides the extra benefit of high precision and localized treatment that generates a strong immunostimulatory effect, suggesting promise for a systemic, immunotherapeutic response. In vitro and in vivo results show that this effect is device-independent and relies largely on operating parameters. No clear cause-effect relationship between specific RONS and the demonstrated immune mediated anticancer efficacy has emerged. Indeed, the same biological outcome (cytotoxicity, innate immune responses, changes in mitochondrial redox potential) can be achieved with two completely different plasma devices that produce a different cocktail of the long-lived RONS. However, these effects cannot be reproduced by applying the same concentrations of long-lived RONS from stable solutions. A second hypothesis is that short-lived species may play a more significant role in the biological outcome. A final hypothesis is that the anti-tumor properties are caused by the change in total cellular redox potential. The goal of this proposal is to investigate whether the effect is total redox-potential based or dependent on the short-lived oxidative species nitric oxide (���������NO), atomic oxygen (O), and the hydroxyl radical (���������OH).

Glioblastoma (GBM) is a common primary brain tumor in adults and carries a dismal prognosis with low survival. The use of conventional chemotherapy is hindered in GBM due to inefficient transport of drugs across the blood brain barrier (BBB). Delivery of chemotherapy to the brain requires either transient disruption of the BBB or local implantation of drug-releasing depots during surgical resection; however, BBB disruption carries health risks while locally-eluting depots are single use and cannot be reused or refilled. Recently, we introduced refillable drug depots, which can be non-invasively refilled through systemic (IV, IP or subQ) administration of inert prodrugs and which release active drugs locally in a sustained manner. Refillable depots have been successful in subcutaneous models of tumor recurrence, preventing tumor growth while eliminating systemic side effects. Unfortunately, the prodrug refills do not cross the BBB and thus refillable depots can not be used in GBM and other brain cancers. We now propose combining refillable drug depots with focused ultrasound (FUS)-induced BBB disruption. FUS-mediated BBB disruption provides a transient (~1 hour) window for the refilling of intracranial depots. The use of nontoxic prodrug refills allows us to administer large doses to maximally exploit the short window for drug delivery. After BBB reformation, the intracranial depots will release active drugs over a period of weeks before being non-invasively refilled again. This innovative combination of these two promising new technologies provides an approach to present therapeutic agents over a long period of time directly to the brain. Our project will further develop this promising approach, optimize parameters, and validate efficacy in preclinical studies. Clinical applications include the local release of chemotherapeutics, biologicals and immunotherapy agents against GBM and other brain cancers. If successful, this approach could have a significant impact in the treatment of a disease which is almost universally lethal currently due to lack of effective treatment strategies. The approach could also be applied to other brain diseases where the BBB presents a challenge to effective treatment.

The objective of this proposal is to obtain funding from the North Carolina Biotechnology Center to purchase a unique, automated, and high throughput slide scanning imager manufactured by Olympus. The new equipment allows for rapid acquisition of highly detailed images of cells and tissues. In biological research, often the generation of large amounts of samples from experiments is desired to tackle important research questions. Yet, sample generation often is not the limiting factor. Rather the imaging and data analysis of large numbers of samples is usually prohibitive and limits the types and impact of research questions that can be asked. As described in this proposal, the Olympus imaging system would unlock a wide range of important biomedical questions in diverse systems from zebrafish to human, bone marrow to brain, by providing an over 10-fold improvement in sample imaging throughput and analysis. The instrument will be installed within an NCSU Shared Core Research Facility, the Cellular and Molecular Imaging Facility (CMIF), which lacks the proposed technology currently, and is staffed by professional staff that will manage, maintain, and operate the proposed equipment. Acquisition of the Olympus slide scanner and installation within CMIF will increase the rate of data acquisition by research groups at NCSU and, in so doing, ensure their continued competitiveness for research funding.

CAR T-cell therapy is a personalized immune-based cancer treatment which harnesses the disease-fighting power of a patient's own immune system by removing T cells from the patient's blood, transforming them in a laboratory to target the cancer, and infusing them back into the patient to attack the cancer. Despite important clinical success, the laboratory transformation takes weeks and costs hundreds of thousands of dollars, severely limiting the therapy������������������s widespread application in cancer treatment. Given the financial burden cancer already poses to the healthcare system, providing this precision health therapeutic to millions of cancer patients presents an insurmountable challenge. In this proposal, we submit feasibility studies for a high-risk high-reward endeavor centered on the hypothesis that the burdensome laboratory process for generating CAR-T cells can be bypassed with use of an implantable scaffold that can recruit T cells, reprogram them into cancer-specific CAR-T cells and release CAR-T cells to the body.