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1. Determining Kinetic Parameters for a Mathematical Model to Optimize Growth of Cancer-Fighting T Cells in a Novel Bioreactor

   Moore, Z (November 2021, Annual Biomedical Research Conference for Minority Students (ABRCMS) Virtual)

   T cell transfer immunotherapy is a highly effective cancer treatment in which the immune system’s inherent ability to fight cancer is amplified by increasing the amount of T cells that are deemed most active within a patient. T cells are a lymphocyte produced as an immune response to cancerous cells. Despite this advanced form of biological therapy, current T cell expansion methods are inefficient, resulting in high manufacturing costs, which brings question to the efficacy of T cell therapies. To address this issue, the recent development of a centrifugal bioreactor aims to rapidly expand T cells for cancer immunotherapy treatments at higher cell densities and in a shorter amount of time compared to current systems on the market. We hypothesize that by producing a mathematical model of a proof-of-concept T cell line to determine substrate consumption and metabolite production over time, we will be able to optimize growth of the cell line in the bioreactor. A series of three studies were performed to produce the growth model: (1) measuring yield coefficients of lactate, ammonium ion, and glucose, (2) determining the Monod constant and maximum specific growth rate, and (3) finding critical metabolite concentrations. To measure yield coefficients, T cells were grown in a 6-well plate at 1 x 105 cells/mL in 4 mL of medium with 100 uL samples taken and frozen each day over a 5-day period. At the end of the study, samples are thawed and used with lactate and ammonium assay kits for microplate reading to determine metabolite levels over time. To determine the Monod constant and maximum specific growth rate, T cells were grown in 12-well plates at pre-calculated varying glucose concentrations in 4 mL of medium in triplicates. Cells were counted for a minimum of six days to determine expansion over time to develop a linearized growth plot. To find critical metabolite concentrations, ammonium and lactate were added to glucose-free T cell medium at four different concentrations in triplicates utilizing a 12-well plate with a seeding density of 1 x 105 cells/mL in 4 mL of medium. The T cells then remained undisturbed in culture and were counted on day three. Once all parameters are determined, we can apply them to the growth model to determine levels of glucose, lactate, and ammonium as the T cells grow to high densities in the bioreactor and, as a result, optimize the manufacturing process for cancer immunotherapy treatments. 

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2. Development and Optimization of a Novel Centrifugal Bioreactor with a Real-Time Monitoring Sensor for T Cell Exhaustion with Applications in Cancer Immunotherapy

   Brenden Fraser-Hevlin, Kitana M. (November 2021, Annual meeting American Institute of Chemical Engineers)

   Cancer has been one of the most significant and critical challenges in the field of medicine. It is a leading cause of death both in the United States and worldwide. Common cancer treatments such as radiation and chemotherapy can be effective in destroying cancerous tissue but cause many detrimental side effects. Thus, recent years have seen new treatment methods that do not harm healthy tissue, including immunotherapy. Adoptive cell therapy (ACT) is one form of immunotherapy in which patients’ immune cells are modified to target cancer cells and then reintroduced into the body. ACT is promising, but most current treatments are inefficient and costly. Widespread implementation of ACT has been a difficult task due to the high treatment cost and inefficient methods currently used to expand the cells. Additionally, if the manufacturing process is not carefully controlled, it can result in the cells losing their cancer-killing ability after expansion. To address the need for an economically feasible culture process to expand immune cells for immunotherapy, our laboratory has designed a centrifugal bioreactor (CBR) expansion system. The CBR uses a balance of centrifugal forces and fluid forces, as shown in Figure 1, to quickly expand infected CD8+ T-cells from a bovine model up to high population densities. With other applications, the CBR has achieved cell densities as high as 1.8 x 108 cells/mL over 7 days in an 11.4-mL chamber. For this study, our goal is to begin validating the CBR by optimizing the growth of CEM (human lymphoblastic leukemia) cells, which are similar cell to cytotoxic T lymphocytes (CTLs). This can be accomplished by measuring kinetic growth parameters based on the concentrations of glucose and inhibitory metabolites in the culture. We hypothesize that by designing a kinetic model from static culture experiments, we can predict the parameters necessary to achieve peak CEM and eventually CTL growth in the CBR. We will report on kinetic growth studies in which different glucose concentrations are tested, and a maximum specific growth rate and Monod constant determined, as well as studies where varying levels of the inhibitory growth byproducts, lactate and ammonium, are added to the culture and critical inhibitor concentrations are determined. Another recent conceptual development for the design of the CBR is a real-time monitoring and feedback control system to regulate the cellular environment, based on levels of surface co-receptors and mRNA signaling within the culture. Prior studies have pinpointed T cell exhaustion as a significant issue in achieving successful immunotherapy, particularly in treatments for solid tumors; T cell exhaustion occurs during a period of chronic antigen stimulation when the cells lose their ability to target and kill cancer cells, currently theorized to be associated with particular inhibitory receptors and cytokines in the immune system. Designing a system with a fiber optic sensor that can monitor the cell state and use feedback control to regulate the pathways involved in producing these receptors will ensure the cells maintain cytotoxic properties during the expansion process within a Centrifugal Fluidized Expansion we call the CentriFLEX. In this presentation, we will also report on early results from development of this exhaustion monitoring system. In brief, achieving optimal kinetic models for the CBR system and methods to prevent T cell exhaustion has the potential to significantly enhance culture efficiency and availability of immunotherapy treatments. 

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3. Chemotherapeutics and CAR‐T Cell‐Based Immunotherapeutics Screening on a 3D Bioprinted Vascularized Breast Tumor Model

   https://doi.org/10.1002/adfm.202203966  

   Dey, Madhuri; Kim, Myoung_Hwan; Dogan, Mikail; Nagamine, Momoka; Kozhaya, Lina; Celik, Nazmiye; Unutmaz, Derya; Ozbolat, Ibrahim_T (October 2022, Advanced Functional Materials)

   Abstract Despite substantial advancements in development of cancer treatments, lack of standardized and physiologically‐relevant in vitro testing platforms limit the early screening of anticancer agents. A major barrier is the complex interplay between the tumor microenvironment and immune response. To tackle this, a dynamic‐flow based 3D bioprinted multi‐scale vascularized breast tumor model, responding to chemo and immunotherapeutics is developed. Heterotypic tumors are precisely bioprinted at pre‐defined distances from a perfused vasculature, exhibit tumor angiogenesis and cancer cell invasion into the perfused vasculature. Bioprinted tumors treated with varying dosages of doxorubicin for 72 h portray a dose‐dependent drug response behavior. More importantly, a cell based immune therapy approach is explored by perfusing HER2‐targeting chimeric antigen receptor (CAR) modified CD8⁺T cells for 24 or 72 h. Extensive CAR‐T cell recruitment to the endothelium, substantial T cell activation and infiltration to the tumor site, resulted in up to ≈70% reduction in tumor volumes. The presented platform paves the way for a robust, precisely fabricated, and physiologically‐relevant tumor model for future translation of anti‐cancer therapies to personalized medicine. 

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4. In Vivo mRNA CAR T Cell Engineering via Targeted Ionizable Lipid Nanoparticles with Extrahepatic Tropism

   https://doi.org/10.1002/smll.202304378  

   Billingsley, Margaret M; Gong, Ningqiang; Mukalel, Alvin J; Thatte, Ajay S; El‐Mayta, Rakan; Patel, Savan K; Metzloff, Ann E; Swingle, Kelsey L; Han, Xuexiang; Xue, Lulu; et al (March 2024, Small)

   Abstract With six therapies approved by the Food and Drug Association, chimeric antigen receptor (CAR) T cells have reshaped cancer immunotherapy. However, these therapies rely on ex vivo viral transduction to induce permanent CAR expression in T cells, which contributes to high production costs and long‐term side effects. Thus, this work aims to develop an in vivo CAR T cell engineering platform to streamline production while using mRNA to induce transient, tunable CAR expression. Specifically, an ionizable lipid nanoparticle (LNP) is utilized as these platforms have demonstrated clinical success in nucleic acid delivery. Though LNPs often accumulate in the liver, the LNP platform used here achieves extrahepatic transfection with enhanced delivery to the spleen, and it is further modified via antibody conjugation (Ab‐LNPs) to target pan‐T cell markers. The in vivo evaluation of these Ab‐LNPs confirms that targeting is necessary for potent T cell transfection. When using these Ab‐LNPs for the delivery of CAR mRNA, antibody and dose‐dependent CAR expression and cytokine release are observed along with B cell depletion of up to 90%. In all, this work conjugates antibodies to LNPs with extrahepatic tropism, evaluates pan‐T cell markers, and develops Ab‐LNPs capable of generating functional CAR T cells in vivo. 

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5. Generation of allogeneic CAR-NKT cells from hematopoietic stem and progenitor cells using a clinically guided culture method

   https://doi.org/10.1038/s41587-024-02226-y  

   Li, Yan-Ruide; Zhou, Yang; Yu, Jiaji; Kim, Yu Jeong; Li, Miao; Lee, Derek; Zhou, Kuangyi; Chen, Yuning; Zhu, Yichen; Wang, Yu-Chen; et al (March 2025, Nature Biotechnology)

   Abstract Cancer immunotherapy with autologous chimeric antigen receptor (CAR) T cells faces challenges in manufacturing and patient selection that could be avoided by using ‘off-the-shelf’ products, such as allogeneic CAR natural killer T (^AlloCAR-NKT) cells. Previously, we reported a system for differentiating human hematopoietic stem and progenitor cells into^AlloCAR-NKT cells, but the use of three-dimensional culture and xenogeneic feeders precluded its clinical application. Here we describe a clinically guided method to differentiate and expand IL-15-enhanced^AlloCAR-NKT cells with high yield and purity. We generated^AlloCAR-NKT cells targeting seven cancers and, in a multiple myeloma model, demonstrated their antitumor efficacy, expansion and persistence. The cells also selectively depleted immunosuppressive cells in the tumor microenviroment and antagonized tumor immune evasion via triple targeting of CAR, TCR and NK receptors. They exhibited a stable hypoimmunogenic phenotype associated with epigenetic and signaling regulation and did not induce detectable graft versus host disease or cytokine release syndrome. These properties of^AlloCAR-NKT cells support their potential for clinical translation. 

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