ENH proofread introduction
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@ -581,14 +581,14 @@ modern manufacturing process.
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The goal of this dissertation was to develop a microcarrier-based \gls{dms} T
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The goal of this dissertation was to develop a microcarrier-based \gls{dms} T
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cell expansion system and determine biologically-meaningful \glspl{cqa} and
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cell expansion system and determine biologically-meaningful \glspl{cqa} and
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\glspl{cpp} that could be used to optimize for highly-potent T cells. In
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\glspl{cpp} that could be used to optimize for highly-potent T cells. In
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\cref{aim1}, we develop and characterized the \gls{dms} system, including
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\cref{aim1}, we developed and characterized the \gls{dms} system, including
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quality control steps. We also demonstrate the feasibility of expanding
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quality control steps. We also demonstrated the feasibility of expanding
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high-quality T cells. In \cref{aim2a,aim2b}, we use \gls{doe} methodology to
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high-quality T cells. In \cref{aim2a,aim2b}, we used \gls{doe} methodology to
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optimize the \gls{dms} platform, and we develop a computational pipeline to
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optimize the \gls{dms} platform, and we developed a computational pipeline to
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identify and model the effects of measurable \glspl{cqa} and \glspl{cpp} on the
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identify and model the effects of measurable \glspl{cqa} and \glspl{cpp} on the
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final product. In \cref{aim3}, we demonstrate the effectiveness of the \gls{dms}
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final product. In \cref{aim3}, we demonstrated the effectiveness of the
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platform \invivo{}. This thesis lays the groundwork for a novel T cell expansion
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\gls{dms} platform \invivo{}. This thesis lays the groundwork for a novel T cell
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method which can be utilized at scale for clinical trials and beyond.
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expansion method which can be utilized at scale for clinical trials and beyond.
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\clearpage
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\clearpage
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@ -604,39 +604,39 @@ diseases\cite{Fesnak2016,Rosenberg2015}. In 2017, Novartis and Kite Pharma
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received FDA approval for \textit{Kymriah} and \textit{Yescarta} respectively,
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received FDA approval for \textit{Kymriah} and \textit{Yescarta} respectively,
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two genetically-modified \gls{car} T cell therapies against B cell malignancies.
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two genetically-modified \gls{car} T cell therapies against B cell malignancies.
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Despite these successes, \gls{car} T cell therapies are constrained by an
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Despite these successes, \gls{car} T cell therapies are constrained by an
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expensive and difficult-to-scale manufacturing process with little control on
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expensive, difficult-to-scale manufacturing process with little control on cell
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cell quality and phenotype\cite{Roddie2019, Dwarshuis2017}. State-of-the-art T
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quality and phenotype\cite{Roddie2019, Dwarshuis2017}. State-of-the-art T cell
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cell manufacturing techniques focus on \acd{3} and \acd{28} activation and
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manufacturing techniques focus on \acd{3} and \acd{28} activation and expansion,
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expansion, typically presented on superparamagnetic, iron-based microbeads
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typically presented on superparamagnetic, iron-based microbeads (Invitrogen
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(Invitrogen Dynabead, Miltenyi MACS beads), on nanobeads (Miltenyi TransACT), or
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Dynabead, Miltenyi MACS beads), on nanobeads (Miltenyi TransACT), or in soluble
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in soluble tetramers (Expamer)\cite{Roddie2019,Dwarshuis2017,Wang2016,
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tetramers (Expamer)\cite{Roddie2019,Dwarshuis2017,Wang2016, Piscopo2017,
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Piscopo2017, Bashour2015}. These strategies overlook many of the signaling
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Bashour2015}. These strategies overlook many of the signaling components
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components present in the secondary lymphoid organs where T cells expand
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present in the secondary lymphoid organs where T cells expand \invivo{}.
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\invivo{}. Typically, T cells are activated under close cell-cell contact, which
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Typically, T cells are activated under close cell-cell contact, which allows for
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allows for efficient autocrine/paracrine signaling via growth-stimulating
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efficient autocrine/paracrine signaling via growth-stimulating cytokines such as
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cytokines such as \gls{il2}. Additionally, the lymphoid tissues are comprised of
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\gls{il2}. Additionally, the lymphoid tissues are comprised of \gls{ecm}
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\gls{ecm} components such as collagen and stromal cells, which provide signals
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components such as collagen and stromal cells, which provide signals to
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to upregulate proliferation, cytokine production, and pro-survival
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upregulate proliferation, cytokine production, and pro-survival
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pathways\cite{Gendron2003, Ohtani2008, Boisvert2007, Ben-Horin2004}.
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pathways\cite{Gendron2003, Ohtani2008, Boisvert2007, Ben-Horin2004}.
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A variety of solutions have been proposed to make the T cell expansion process
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A variety of solutions have been proposed to make the T cell expansion process
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more physiological. Including feeder cell cultures\cite{Forget2014} and
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more physiological. These include feeder cell cultures\cite{Forget2014} and
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biomaterials-based methods such as lipid-coated microrods or 3D scaffold
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biomaterials-based methods such as lipid-coated microrods or 3D scaffold
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gels\cite{Cheung2018,Delalat2017,meyer15_immun,Lambert2017} that attempt to
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gels\cite{Cheung2018,Delalat2017,meyer15_immun,Lambert2017} that attempt to
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recapitulate the cellular membrane, large interfacial contact area,
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recapitulate the cellular membrane, large interfacial contact area,
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3D-structure, or soft surfaces T cells normally experience \invivo{}. While
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3D-structure, or soft surfaces T cells normally experience \invivo{}. While
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these have been shown to activation and expand T cells, they either are not
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these have been shown to activate and expand T cells, they either are not
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scalable (in the case of feeder cells) or still lack many of the signals and
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scalable (in the case of feeder cells) or still lack many of the signals and
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cues T cells experience as the expand. Additionally, none have been shown to
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cues T cells experience as the expand. Additionally, none have been shown to
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preferentially expand highly-potent T cell necessary for anti-cancer therapies.
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preferentially expand highly-potent T cell necessary for anti-cancer therapies.
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Such high potency cells including subtypes with low differentiation state such
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Such high potency cells are subtypes with low differentiation state such
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as \gls{tscm} and \gls{tcm} cells or CD4 cells, all of which have been shown to
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as \gls{tscm} and \gls{tcm} cells or CD4 cells, all of which have been shown to
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be necessary for durable responses\cite{Xu2014, Fraietta2018, Gattinoni2011,
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be necessary for durable responses\cite{Xu2014, Fraietta2018, Gattinoni2011,
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Gattinoni2012,Wang2018, Yang2017}. Methods to increase memory and CD4 T cells
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Gattinoni2012,Wang2018, Yang2017}. Methods to increase memory and CD4 T cells
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in the final product are needed. Furthermore, \gls{qbd} principles such as
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in the final product are needed. Furthermore, \gls{qbd} principles such as
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discovering and validating novel \glspl{cqa} and \glspl{cpp} in the space of T
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discovering and validating novel \glspl{cqa} and \glspl{cpp} in the space of T
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cell manufacturing are required to reproducibly manufacture these subtypes and
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cell manufacturing are required to reproducibly manufacture these subtypes and
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ensure low-cost and safe products with maximal effectiveness in the clinic
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ensure low-cost and safe products with maximal effectiveness in the clinic.
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This dissertation describes a novel \acrlong{dms}-based method derived from
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This dissertation describes a novel \acrlong{dms}-based method derived from
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porous microcarriers functionalized with \acd{3} and \acd{28} \glspl{mab} for
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porous microcarriers functionalized with \acd{3} and \acd{28} \glspl{mab} for
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@ -654,11 +654,11 @@ emulate the large contact surface area that occurs between T cells and
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\section*{hypothesis}
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\section*{hypothesis}
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The hypothesis of this dissertation was that using \glspl{dms} created from
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The hypothesis of this dissertation was that using \glspl{dms} created from
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off-the-shelf microcarriers and coated with activating \glspl{mab} would lead to
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off-the-shelf microcarriers and coated with activating \glspl{mab} would
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higher quantity and quality T cells as compared to state-of-the-art bead-based
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increase quantity and quality of T cells as compared to state-of-the-art
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expansion. We also hypothesized that T cells have measurable biological
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bead-based expansion. We also hypothesized that such T cells have measurable
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signatures that are predictive of downstream outcomes and phenotypes. The
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biological signatures that are predictive of downstream outcomes and phenotypes.
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objective of this dissertation was to develop this platform, test its
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The objective of this dissertation was to develop this platform, test its
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effectiveness both \invitro{} and \invivo{}, and develop computational pipelines
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effectiveness both \invitro{} and \invivo{}, and develop computational pipelines
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to discover novel \glspl{cpp} and \glspl{cqa} that can be translated to a
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to discover novel \glspl{cpp} and \glspl{cqa} that can be translated to a
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manufacturing environment and a clinical trial setting.
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manufacturing environment and a clinical trial setting.
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@ -683,10 +683,10 @@ The specific aims of this dissertation are outlined in
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In this first aim, we demonstrated the process for manufacturing \glspl{dms},
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In this first aim, we demonstrated the process for manufacturing \glspl{dms},
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including quality control steps that are necessary for translation of this
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including quality control steps that are necessary for translation of this
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platform into a scalable manufacturing setting. We also demonstrate that the
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platform into a scalable manufacturing setting. We also demonstrated that the
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\gls{dms} platform leads to higher overall expansion of T cells and higher
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\gls{dms} platform leads to higher overall expansion of T cells and higher
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overall fractions of potent memory and CD4+ subtypes desired for T cell
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overall fractions of potent memory and CD4+ subtypes desired for T cell
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therapies. Finally, we demonstrate \invitro{} that the \gls{dms} platform can be
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therapies. Finally, we showed \invitro{} that the \gls{dms} platform can be
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used to generate functional \gls{car} T cells targeted toward CD19.
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used to generate functional \gls{car} T cells targeted toward CD19.
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\subsection*{aim 2: develop methods to control and predict T cell quality}
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\subsection*{aim 2: develop methods to control and predict T cell quality}
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@ -705,7 +705,7 @@ accomplished through two sub-aims:
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\subsection*{aim 3: confirm potency of T cells from novel T cell expansion
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\subsection*{aim 3: confirm potency of T cells from novel T cell expansion
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process using \invivo{} xenograft mouse model}
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process using \invivo{} xenograft mouse model}
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In this final aim, we demonstrate the effectiveness of \gls{dms}-expanded T
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In this final aim, we demonstrated the effectiveness of \gls{dms}-expanded T
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cells compared to state-of-the-art beads using \invivo{} mouse models for
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cells compared to state-of-the-art beads using \invivo{} mouse models for
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\gls{all}.
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\gls{all}.
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@ -732,27 +732,28 @@ manipulation\cite{Kirouac2008, Little2006, Pirnay2012, Rousseau2013}. This is
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further compounded by the lack of standardization and limited regulation.
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further compounded by the lack of standardization and limited regulation.
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In order to overcome these barriers, adopting a systemic approach to cell
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In order to overcome these barriers, adopting a systemic approach to cell
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manufacturing using \acrlong{qbd} principles will be extremely
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manufacturing using \acrfull{qbd} principles will be extremely
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important\cite{Kirouac2008}. In \gls{qbd}, the objective is to reproducibly
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important\cite{Kirouac2008}. In \gls{qbd}, the objective is to reproducibly
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manufacturing products which minimizes risk for downstream stakeholders (in this
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manufacturing products which minimizes risk for downstream stakeholders (in this
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case, the patient). Broadly, this entails determining \acrlongpl{cqa} and
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case, the patient). This entails determining \acrlongpl{cqa} and \acrlongpl{cpp}
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\acrlongpl{cpp} and incorporating them into models which can explain and predict
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and incorporating them into models which can explain and predict the cell
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the cell manufacturing process.
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manufacturing process.
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\Glspl{cqa} are measurable properties of the product that can be used to define
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\Glspl{cqa} are measurable properties of the product that are used to define its
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its functionality and hence quality. \glspl{cqa} are important for defining the
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functionality and hence quality. \glspl{cqa} are important for defining the
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characteristics of a `good' product (release criteria) but also for ensuring
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characteristics of a ``good'' product (release criteria) but also for ensuring
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that a process is on track to making such a product (process control). In the
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that a process is on track to making such a product (process control). In the
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space of cell manufacturing, examples of \glspl{cqa} include markers on the
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space of cell manufacturing, examples of \glspl{cqa} include markers on the
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surface of cells and readouts from functional assays such as killing assays. In
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surface of cells and readouts from functional assays such as killing assays. In
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general, these are poorly understood if they exist at all.
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general, these are poorly understood if they exist at all.
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%% TODO IL2 use here is wonky
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\glspl{cpp} are parameters which may be tuned and varied to control the outcome
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\glspl{cpp} are parameters which may be tuned and varied to control the outcome
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of process and the quality of the final product. In cell manufacturing, these
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of process and the quality of the final product. Examples include the type of
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are poorly understood. Examples in the cell manufacturing space include the type
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media used and the amount of \il{2} added. While these can be easy to control,
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of media used and the amount of \il{2} added. Once \glspl{cpp} are known, they
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the effect they have on the final outcome is generally unknown. Once \glspl{cpp}
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can be optimized to ensure that costs are minimized and potency of the cellular
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are known, they can be optimized to ensure that costs are minimized and potency
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product is maximized.
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of the cellular product is maximized.
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The topic of discovering novel \glspl{cpp} and \glspl{cqa} in the context of
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The topic of discovering novel \glspl{cpp} and \glspl{cqa} in the context of
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this work are discussed further in \cref{sec:background_doe} and
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this work are discussed further in \cref{sec:background_doe} and
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@ -767,12 +768,13 @@ theory the technology developed in this dissertation could theoretically apply
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to any T cell-based therapy with little to no modification.
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to any T cell-based therapy with little to no modification.
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One of the first successful T cell-based immunotherapies against cancer is
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One of the first successful T cell-based immunotherapies against cancer is
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\glspl{til}\cite{Rosenberg2015}. This method works by taking tumor specimens
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\glspl{til}\cite{Rosenberg2015}. This method works by excising tumor fragments
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from a patient, allowing the tumor-reactive lymphocytes to expand \exvivo{}, and
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from a patient, allowing the tumor-reactive lymphocytes to expand \exvivo{} from
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then administered back to the patient along with a high dose of
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within these fragments, and then administered these lymphocytes back to the
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\il{2}\cite{Rosenberg1988}. In particular, \gls{til} therapy has shown robust
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patient along with a high dose of \il{2}\cite{Rosenberg1988}. In particular,
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results in treating melanoma\cite{Rosenberg2011}, although \glspl{til} have been
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\gls{til} therapy has shown robust results in treating
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found in other solid tumors such as gastointestinal, cervical, lung, and
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melanoma\cite{Rosenberg2011}, although \glspl{til} have been found in other
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solid tumors such as gastointestinal, cervical, lung, and
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ovarian\cite{Rosenberg2015, Wang2014, Foppen2015, Solinas2017, June2007,
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ovarian\cite{Rosenberg2015, Wang2014, Foppen2015, Solinas2017, June2007,
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Santoiemma2015}, and their presence is generally associate with favorable
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Santoiemma2015}, and their presence is generally associate with favorable
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outcomes\cite{Clark1989}. \glspl{til} are heterogeneous cell mixtures and
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outcomes\cite{Clark1989}. \glspl{til} are heterogeneous cell mixtures and
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@ -795,26 +797,27 @@ T cells with transduced \glspl{tcr} were first designed to overcome the
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limitations of \glspl{til}\cite{Rosenberg2015, Wang2014}. In this case, T cells
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limitations of \glspl{til}\cite{Rosenberg2015, Wang2014}. In this case, T cells
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are transduced \exvivo{} with a lentiviral vector to express a \gls{tcr}
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are transduced \exvivo{} with a lentiviral vector to express a \gls{tcr}
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targeting a tumor antigen. T cells transduced with \glspl{tcr} have shown robust
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targeting a tumor antigen. T cells transduced with \glspl{tcr} have shown robust
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results in melanoma patients\cite{Robbins2011}, synovial
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results against melanoma \cite{Robbins2011}, synovial sarcoma\cite{Morgan2006},
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sarcoma\cite{Morgan2006}, and others\cite{Ikeda2016}. To date, there are over
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and others\cite{Ikeda2016}. To date, there are over 200 clinical trials using T
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200 clinical trials using T cells with transduced \glspl{tcr}.
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cells with transduced \glspl{tcr}.
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While transduced \glspl{tcr} offer some flexibility in retargeting T cells
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While transduced \glspl{tcr} offer some flexibility in retargeting T cells
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toward relevant tumor antigens, they are still limited in that they can only
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toward relevant tumor antigens, they are still limited in that they can only
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target antigens that are presented via \gls{mhc} complexes. \acrlong{car} T
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target antigens that are presented via \gls{mhc}. \Acrlong{car} T cells overcome
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cells overcome this limitation by using linking a \gls{tcr}-independent antigen
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this limitation by linking a \gls{tcr}-independent antigen recognition domain
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recognition domain with the stimulatory and costimulatory machinery of a T cell
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with the stimulatory and costimulatory machinery of a T cell. \gls{car} T cells
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\gls{car} T cells were first demonstrated in 1989, where the authors swapped the
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were first demonstrated in 1989, where the authors swapped the
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antigen-recognition domains of a native \gls{tcr} with a that of a foreign
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antigen-recognition domains of a native \gls{tcr} with a that of a foreign
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\gls{tcr}\cite{Gross1989}. Since then, this method has progressed to using an
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\gls{tcr}\cite{Gross1989}. Since then, this method has progressed to using an
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\gls{scfv} with a CD3$\upzeta$ stimulatory domain along with the CD28, OX-40, or
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\gls{scfv} for antigen recognition, a CD3$\upzeta$ domain for the stimulatory
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4-1BB domains for costimulation. Since these can all be expressed with one
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signal, and a CD28, OX-40, or 4-1BB domains for the costimulatory signal. Since
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protein sequence, \gls{car} T cells are relatively simple to produce and require
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these can all be expressed with one protein sequence, \gls{car} T cells are
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only a single genetic transduction step (usually a lentiviral vector) to
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relatively simple to produce and require only a single genetic transduction step
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reprogram a batch T cells \exvivo{} toward the desired antigen. \gls{car} T
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(usually a lentiviral vector) to reprogram a batch T cells \exvivo{} toward the
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cells have primarily found success in against CD19- and CD20-expressing tumors
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desired antigen. \gls{car} T cells have primarily found success in against CD19-
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such as \gls{all} and \gls{cll} (eg B-cell malignancies)\cite{Kalos2011,
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and CD20-expressing tumors such as \gls{all} and \gls{cll} (eg B-cell
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Brentjens2011, Kochenderfer2010, Maude2014, Till2012, Till2008}.
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malignancies)\cite{Kalos2011, Brentjens2011, Kochenderfer2010, Maude2014,
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Till2012, Till2008}.
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Out of all the T cell therapies discussed thus far, \gls{car} T cells have
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Out of all the T cell therapies discussed thus far, \gls{car} T cells have
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experienced the most commercial success and excitement. In 2017, Novartis and
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experienced the most commercial success and excitement. In 2017, Novartis and
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@ -828,9 +831,8 @@ date, there are almost 1000 clinical trials using \gls{car} T cells.
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\subsection{Scaling T Cell Expansion}
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\subsection{Scaling T Cell Expansion}
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In order to scale T cell therapies to meet clinical demands, automation and
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In order to scale T cell therapies, automation and bioreactors will be
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bioreactors will be necessary. To this end, there are several choices that have
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necessary. To this end, several choices have found success in the clinic.
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found success in the clinic.
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The WAVE bioreactor (GE Healthcare) is the choice of expansion for many clinical
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The WAVE bioreactor (GE Healthcare) is the choice of expansion for many clinical
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applications\cite{Brentjens2011, Hollyman2009, Brentjens2013}. It is part of a
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applications\cite{Brentjens2011, Hollyman2009, Brentjens2013}. It is part of a
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@ -844,32 +846,30 @@ design, which could have negative impact on cross-talk, activation, and
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growth\cite{Somerville2012}.
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growth\cite{Somerville2012}.
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% BACKGROUND find clinical trials (if any) that use this
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% BACKGROUND find clinical trials (if any) that use this
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Alternatively, the CliniMACS Prodigy (Miltenyi) is an all-in-one system that is
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Alternatively, the CliniMACS Prodigy (Miltenyi) is an all-in-one, fully-closed
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a fully closed system that removes the need for expensive cleanrooms and
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system that removes the need for expensive cleanrooms and associated
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associated personnel\cite{Kaiser2015, Bunos2015}. It contains modules to perform
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personnel\cite{Kaiser2015, Bunos2015}. It contains modules to perform
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transduction, expansion, and washing. This setup also implies that fewer
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transduction, expansion, and washing. This setup is less prone to mistakes,
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mistakes and handling errors will be made, since many of the steps are internal
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since most steps are internal to the machine. Initial investigations have shown
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to the machine. Initial investigations have shown that it can yield T cells
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that it can yield T cells doses required for clinical use\cite{Zhu2018}. At the
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doses required for clinical use\cite{Zhu2018}. At the time of writing, several
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time of writing, several clinical trial are underway which use the CliniMACS,
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clinical trial are underway which use the CliniMACS, although mostly for
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although mostly for stem-cell based cell treatments.
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stem-cell based cell treatments.
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Finally, another option that has been investigated for T cell expansion is the
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Finally, another option that has been investigated for T cell expansion is the
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Grex bioreactor (Wilson Wolf). This is effectively a tall tissue-culture plate
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Grex bioreactor (Wilson Wolf). This is effectively a tall tissue-culture plate
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with a porous membrane at the bottom, which allows gas exchange to the active
|
with a porous membrane at the bottom. This allows large volumes of media to be
|
||||||
cell culture at the bottom of the plate while permitting large volumes of media
|
loaded without suffocating the cells, which can exchange gas through the
|
||||||
to be loaded on top without suffocating the cells. While this is quite similar
|
membrane. While this is quite similar to plates and flasks normally used for
|
||||||
to plates and flasks normally used for small-scale research, the important
|
small-scale research, the important difference is that its larger size requires
|
||||||
difference is that its larger size requires fewer interactions and keeps the
|
fewer interactions and keeps the cells at a higher nutrient concentration for
|
||||||
cells at a higher nutrient concentration for longer periods of time. However, it
|
longer periods of time. However, it is still a an open system and requires
|
||||||
is still a an open system and requires manual (by default) interaction from an
|
manual (by default) interaction from an operator to load, feed, and harvest the
|
||||||
operator to load, feed, and harvest the cell product. Grex bioreactors have been
|
cell product. Grex bioreactors have been using to grow \glspl{til}\cite{Jin2012}
|
||||||
using to grow \glspl{til}\cite{Jin2012} and virus-specific T
|
and virus-specific T cells\cite{Gerdemann2011}.
|
||||||
cells\cite{Gerdemann2011}.
|
|
||||||
|
|
||||||
Much work is still required in the space of bioreactor design for T cell
|
Much work is still required in the space of bioreactor design for T cell
|
||||||
manufacturing, but novel T cell expansion technologies such as that described in
|
manufacturing, but novel T cell expansion technologies such as that described in
|
||||||
this work need to consider how it may be used at scale in such a system.
|
this work need to consider how they may be used at scale in such a system.
|
||||||
|
|
||||||
\subsection{Cell Sources in T Cell Manufacturing}\label{sec:background_source}
|
\subsection{Cell Sources in T Cell Manufacturing}\label{sec:background_source}
|
||||||
|
|
||||||
|
@ -887,35 +887,34 @@ cross-reactivity with the patient and thus \gls{gvhd} is not a
|
||||||
concern\cite{Decker2012}. However, there are numerous disadvantages. Autologous
|
concern\cite{Decker2012}. However, there are numerous disadvantages. Autologous
|
||||||
therapies are over 20 times more costly as the process needs to be repeated for
|
therapies are over 20 times more costly as the process needs to be repeated for
|
||||||
every patient\cite{Harrison2019}. Compounding this, many cell products are
|
every patient\cite{Harrison2019}. Compounding this, many cell products are
|
||||||
manufactured at a centralized location, so patient T cells need to be shipped
|
manufactured at a centralized location, so cells need to be shipped on dry ice
|
||||||
twice on dry ice from the hospital and back. This adds days to the process,
|
from the hospital and back. This adds days to the process, which is critical for
|
||||||
which is critical for patients with fast moving diseases. Manufacturing could be
|
patients with fast moving diseases. Manufacturing could be done on-site in a
|
||||||
done on-site in a decentralized manner, but this requires more equipment and
|
decentralized manner, but this requires more equipment and personnel overall.
|
||||||
personnel overall. Using cells from a diseased patient has many drawbacks in
|
Sourcing cells from a diseased patient has many drawbacks in itself. Cancer
|
||||||
itself. Cancer patients (especially those with chronic illnesses) often have
|
patients (especially those with chronic illnesses) often have exhausted T cells
|
||||||
exhausted T cells which expand far less readily and are consequently less
|
which expand far less readily and are consequently less potent\cite{Wherry2015,
|
||||||
potent\cite{Wherry2015, Ando2019, Zheng2017}. Additionally, they may have high
|
Ando2019, Zheng2017}. Additionally, they may have high frequencies of
|
||||||
frequencies of \glspl{treg} which inhibitory\cite{Decker2012}. Removing these
|
\glspl{treg} which have an inhibitory effect on
|
||||||
cells as well as purifying \glspl{th1} may enhance the potency of the final
|
immunotherapies\cite{Decker2012}. Removing these cells as well as purifying
|
||||||
product\cite{Goldstein2012, Drela2004, Rankin2003, Luheshi2013, Grotz2015};
|
\glspl{th1} may enhance the potency of the final product\cite{Goldstein2012,
|
||||||
however, this would make the overall process more expensive as an additional
|
Drela2004, Rankin2003, Luheshi2013, Grotz2015}; however, this makes the
|
||||||
separation step would be required.
|
overall process more expensive as an additional separation step is required.
|
||||||
|
|
||||||
Allogeneic T cell therapies overcome nearly all of these disadvantages. Donor
|
Allogeneic T cell therapies overcome nearly all of these disadvantages. Donor
|
||||||
\glspl{pbmc} are easy to obtain, they can be processed in centralized locations,
|
\glspl{pbmc} are easy to obtain, they can be processed in centralized locations,
|
||||||
they can be stored easily under liquid nitrogen, and donors could be screened to
|
and they can be stored easily under liquid nitrogen. Donors can also be screened
|
||||||
find those with optimal anti-tumor cells. The key is overcoming \gls{gvhd}.
|
to find those with optimal anti-tumor cells. The key is overcoming \gls{gvhd}.
|
||||||
Obviously this could be done the same way as done for transplants where patients
|
Obviously this could be done analogously to transplants where patients find a
|
||||||
find a `match' for their \gls{hla} type, but this severally limits options. This
|
``match'' for their \gls{hla} type, but this severally limits options. This can
|
||||||
can be overcome by using advanced gene-editing tools which can both add and
|
be overcome by using advanced gene-editing tools (eg \glspl{zfn}, \glspl{talen},
|
||||||
delete genetic material (eg \glspl{zfn}, \glspl{talen}, or \gls{crispr}) to
|
or \gls{crispr}) to remove the native \gls{tcr} and thus prevent the donor T
|
||||||
remove the native \gls{tcr} which would prevent the donor T cells from attacking
|
cells from attacking host tissue\cite{Liu2019, Wiebking2020, Provasi2012,
|
||||||
host tissue\cite{Liu2019, Wiebking2020, Provasi2012, Berdien2014, Themeli2015}.
|
Berdien2014, Themeli2015}. This obviously complicates the process, as
|
||||||
This obviously complicates the process, as additional edits besides the
|
additional edits besides the insertion of the \gls{car} would be required, and
|
||||||
insertion of the \gls{car} would be required, and these technologies are not yet
|
these technologies are not yet very efficient. To date there are about 10 open
|
||||||
very efficient. To date there are about 10 open clinical trials utilizing
|
clinical trials utilizing allogeneic T cell therapies edited with \gls{crispr}
|
||||||
allogeneic T cell therapies edited with \gls{crispr} to reduce the likelihood of
|
to reduce the likelihood of \gls{gvhd}.
|
||||||
\gls{gvhd}.
|
|
||||||
|
|
||||||
\subsection{Overview of T Cell Quality}\label{sec:background_quality}
|
\subsection{Overview of T Cell Quality}\label{sec:background_quality}
|
||||||
|
|
||||||
|
@ -927,13 +926,13 @@ criteria, and initial cell source screening.
|
||||||
|
|
||||||
One of the most important dimensions of T cell quality is that of
|
One of the most important dimensions of T cell quality is that of
|
||||||
differentiation. T cells begin their life in circulation (eg after they exit the
|
differentiation. T cells begin their life in circulation (eg after they exit the
|
||||||
thymus) as \glspl{tn}. When they become activated in the secondary lymph node
|
thymus) as \glspl{tn}. When they become activated in the secondary lymphoid
|
||||||
organs, they differentiate from \gls{tn} to \glspl{tscm}, \glspl{tcm},
|
organs, they differentiate from \gls{tn} to \glspl{tscm}, \glspl{tcm},
|
||||||
\glspl{tem}, and finally \glspl{teff}\cite{Gattinoni2012}. Subtypes earlier in
|
\glspl{tem}, and finally \glspl{teff}\cite{Gattinoni2012}. Subtypes earlier in
|
||||||
this process are generally called `memory' or `memory-like' cells (eg \gls{tscm}
|
this process are generally called ``memory'' or ``memory-like'' cells (eg
|
||||||
and \gls{tcm}), and have been shown to have increased potency toward a variety
|
\gls{tscm} and \gls{tcm}), and have been shown to have increased potency toward
|
||||||
of tumors, presumably due to their higher capacity for self-renewal and
|
a variety of tumors, presumably due to their higher capacity for self-renewal
|
||||||
replication, enhanced migratory capacity, and/or increased engraftment
|
and replication, enhanced migratory capacity, and/or increased engraftment
|
||||||
potential\cite{Xu2014, Gattinoni2012, Fraietta2018, Gattinoni2011, Turtle2009}.
|
potential\cite{Xu2014, Gattinoni2012, Fraietta2018, Gattinoni2011, Turtle2009}.
|
||||||
The capacity for self-renewal is especially important for T cells therapies, as
|
The capacity for self-renewal is especially important for T cells therapies, as
|
||||||
evidenced by the fact that \gls{til} therapies with longer telomeres tend to
|
evidenced by the fact that \gls{til} therapies with longer telomeres tend to
|
||||||
|
@ -951,7 +950,7 @@ In addition to memory, the other major axis by which T cells may be classified
|
||||||
is the CD4/CD8 ratio. \Glspl{th} are CD4+ are responsible for secreting
|
is the CD4/CD8 ratio. \Glspl{th} are CD4+ are responsible for secreting
|
||||||
cytokines which coordinate the immune response while CD8+ \glspl{tc} are
|
cytokines which coordinate the immune response while CD8+ \glspl{tc} are
|
||||||
responsible for killing tumors or infected cells using specialized lytic
|
responsible for killing tumors or infected cells using specialized lytic
|
||||||
enzymes. Since \glspl{tc} actually perform the killing function, it seems
|
enzymes. Since \glspl{tc} actually possess the killing function, it seems
|
||||||
intuitive that \glspl{tc} would be most important for anti-tumor
|
intuitive that \glspl{tc} would be most important for anti-tumor
|
||||||
immunotherapies. However, in mouse models with glioblastoma, survival was
|
immunotherapies. However, in mouse models with glioblastoma, survival was
|
||||||
negatively impacted when \glspl{th} were removed\cite{Wang2018}. Furthermore,
|
negatively impacted when \glspl{th} were removed\cite{Wang2018}. Furthermore,
|
||||||
|
@ -969,10 +968,10 @@ radioactive tracer, by measuring the degranulation of the T cells themselves, or
|
||||||
by measuring a cytokine that is secreted upon T cell activation and killing such
|
by measuring a cytokine that is secreted upon T cell activation and killing such
|
||||||
as \gls{ifng}. Furthermore, the viability of T cells may be assessed using a
|
as \gls{ifng}. Furthermore, the viability of T cells may be assessed using a
|
||||||
number of methods, including exclusion dyes such as \gls{aopi} or a functional
|
number of methods, including exclusion dyes such as \gls{aopi} or a functional
|
||||||
assay to detect metabolism. Finally, for the purposes of safety, T cell products
|
assay to detect metabolism. Finally, for safety, retro- or lentivirally
|
||||||
using retro- or lentiviral vectors as their means of gene-editing must be tested
|
transduced T cell products must be tested for replication competent
|
||||||
for replication competent vectors\cite{Wang2013} and for contamination via
|
vectors\cite{Wang2013}, and all cell products in general should be tested for
|
||||||
bacteria or other pathogens.
|
bacterial or fungal contamination.
|
||||||
|
|
||||||
\subsection{T Cell Activation Methods}\label{sec:background_activation}
|
\subsection{T Cell Activation Methods}\label{sec:background_activation}
|
||||||
|
|
||||||
|
@ -983,24 +982,23 @@ present a peptide via \gls{mhc} that the T cell in question is able to
|
||||||
recognize. Signal 2 is administered via CD80 and CD86 which are also present on
|
recognize. Signal 2 is administered via CD80 and CD86 which are also present on
|
||||||
\glspl{apc} and is necessary to prevent the T cell from becoming anergic. While
|
\glspl{apc} and is necessary to prevent the T cell from becoming anergic. While
|
||||||
these two signal are the bare minimum to trigger a T cell to expand, there are
|
these two signal are the bare minimum to trigger a T cell to expand, there are
|
||||||
many other potential signals present. T cells have many other costimulatory
|
many other potential signals present. T cells have other receptors such as OX40,
|
||||||
receptors such as OX40, 4-1BB and ICOS which are costimulatory along with CD28,
|
4-1BB, and ICOS which are costimulatory along with CD28, and \glspl{apc} have
|
||||||
and \glspl{apc} have corresponding ligands for these depending on the nature of
|
corresponding ligands for these depending on the nature of the pathogen they
|
||||||
the pathogen they have detected\cite{Azuma2019}. Furthermore, T cells exist in
|
have detected\cite{Azuma2019}. Furthermore, T cells exist in high cell density
|
||||||
high cell density within the secondary lymphoid organs, which allows efficient
|
within the secondary lymphoid organs, which allows efficient cytokine cross-talk
|
||||||
cytokine cross-talk in an autocrine and paracrine manner. These cytokines are
|
in an autocrine and paracrine manner. These cytokines are responsible for
|
||||||
responsible for triggering proliferation (in the case of \il{2}) and subset
|
triggering proliferation (in the case of \il{2}) and subset differentiation (in
|
||||||
differentiation (in the case of many others)\cite{Luckheeram2012}. By tuning the
|
the case of many others)\cite{Luckheeram2012}. By tuning the signal strength and
|
||||||
signal strength and duration of Signal 1, Signal 2, the various costimulatory
|
duration of Signal 1, Signal 2, the various costimulatory signals, and the
|
||||||
signals, and the cytokine milieu, a variety of T cell phenotypes can be
|
cytokine milieu, a variety of T cell phenotypes can be actualized.
|
||||||
actualized.
|
|
||||||
|
|
||||||
\Invitro{}, T cells can be activated in a number of ways but the simplest and
|
There are many ways to activate T cells \invitro{}, but the simplest and most
|
||||||
most common is to use \glspl{mab} that cross-link the CD3 and CD28 receptors,
|
common is to use \glspl{mab} that cross-link CD3 and CD28, which supply Signal 1
|
||||||
which supply Signal 1 and Signal 2 without the need for antigen (which also
|
and Signal 2 without the need for antigen (which also means all T cells are
|
||||||
means all T cells are activated and not just a few specific clones). Additional
|
activated and not just a few specific clones). Additional signals may be
|
||||||
signals may be supplied in the form of cytokines (eg \il{2}, \il{7}, or \il{15})
|
supplied in the form of cytokines (eg \il{2}, \il{7}, or \il{15}) or feeder
|
||||||
or feeder cells\cite{Forget2014}.
|
cells\cite{Forget2014}.
|
||||||
|
|
||||||
As this is a critical unit operation in the manufacturing of T cell therapies, a
|
As this is a critical unit operation in the manufacturing of T cell therapies, a
|
||||||
number of commercial technologies exist to activate T cells\cite{Wang2016,
|
number of commercial technologies exist to activate T cells\cite{Wang2016,
|
||||||
|
@ -1009,11 +1007,11 @@ number of commercial technologies exist to activate T cells\cite{Wang2016,
|
||||||
accomplished in a \gls{gmp} manner (at least from a reagents perspective) as
|
accomplished in a \gls{gmp} manner (at least from a reagents perspective) as
|
||||||
soluble \gls{gmp}-grade \glspl{mab} are commericially available. A similar but
|
soluble \gls{gmp}-grade \glspl{mab} are commericially available. A similar but
|
||||||
distinct method along these lines is to use multivalent activators such as
|
distinct method along these lines is to use multivalent activators such as
|
||||||
ImmunoCult (StemCell Technologies) or Expamer (Juno Therapeutics) which may have
|
ImmunoCult (StemCell Technologies) or Expamer (Juno Therapeutics) which have
|
||||||
increased cross-linking capacity compared to traditional \glspl{mab}. Beyond
|
increased cross-linking capacity compared to traditional \glspl{mab}. Beyond
|
||||||
soluble protein, \glspl{mab} against CD3 and CD28 can be mounted on magnetic
|
soluble protein, \glspl{mab} against CD3 and CD28 can be mounted on magnetic
|
||||||
microbeads (\SIrange{3}{5}{\um} in diameter) such as DynaBeads (Invitrogen) and
|
microbeads (\SIrange{3}{5}{\um} in diameter) such as DynaBeads (Invitrogen) and
|
||||||
MACSbeads (\miltenyi{}), which are easier to separate using magnetic washing
|
MACSbeads (\miltenyi{}), which are easy to separate using magnetic washing
|
||||||
plates. Magnetic nanobeads such as TransAct (\miltenyi{}) work by a similar
|
plates. Magnetic nanobeads such as TransAct (\miltenyi{}) work by a similar
|
||||||
principle except they can be removed via centrifugation rather than a magnetic
|
principle except they can be removed via centrifugation rather than a magnetic
|
||||||
washing plate. Cloudz (RnD Systems) are another bead-based T cell expansion
|
washing plate. Cloudz (RnD Systems) are another bead-based T cell expansion
|
||||||
|
@ -1026,18 +1024,17 @@ Signal 1 and Signal 2 and ignore the many other physiological cues present in
|
||||||
the secondary lymphoid organs. A variety of novel T cell activation and
|
the secondary lymphoid organs. A variety of novel T cell activation and
|
||||||
expansion solutions have been proposed to overcome this. One strategy is to use
|
expansion solutions have been proposed to overcome this. One strategy is to use
|
||||||
modified feeder cell cultures to provide activation signals similar to those of
|
modified feeder cell cultures to provide activation signals similar to those of
|
||||||
\glspl{dc}\cite{Forget2014}. While this has the theoretical capacity to mimic
|
\glspl{dc}\cite{Forget2014}. While this can theoretically mimic many components
|
||||||
several key components of the lymph node, it is hard to reproduce on a large
|
of the lymph node, it is hard to scale due to the complexity and inherent
|
||||||
scale due to the complexity and inherent variability of using cell lines in a
|
variability of using cell lines in a \gls{gmp}-compliant manner. Others have
|
||||||
fully \gls{gmp}-compliant manner. Others have proposed biomaterials-based
|
proposed biomaterials-based solutions to circumvent this problem, including
|
||||||
solutions to circumvent this problem, including lipid-coated
|
lipid-coated microrods\cite{Cheung2018}, 3D-scaffolds via either
|
||||||
microrods\cite{Cheung2018}, 3D-scaffolds via either Matrigel\cite{Rio2018} or
|
Matrigel\cite{Rio2018} or 3d-printed lattices\cite{Delalat2017}, ellipsoid
|
||||||
3d-printed lattices\cite{Delalat2017}, ellipsoid beads\cite{meyer15_immun}, and
|
beads\cite{meyer15_immun}, and \gls{mab}-conjugated \gls{pdms}
|
||||||
\gls{mab}-conjugated \gls{pdms} beads\cite{Lambert2017} that respectively
|
beads\cite{Lambert2017} that respectively recapitulate the cellular membrane,
|
||||||
recapitulate the cellular membrane, large interfacial contact area,
|
large interfacial contact area, 3D-structure, or soft surfaces T cells normally
|
||||||
3D-structure, or soft surfaces T cells normally experience \textit{in vivo}.
|
experience \textit{in vivo}. None of these have been shown to expand high
|
||||||
None have been demonstrated to demonstrably expand high quality T cells as
|
quality T cells as outlined in \cref{sec:background_quality}.
|
||||||
outlined in \cref{sec:background_quality}.
|
|
||||||
|
|
||||||
\subsection{Microcarriers in Bioprocessing}
|
\subsection{Microcarriers in Bioprocessing}
|
||||||
|
|
||||||
|
@ -1046,56 +1043,54 @@ methods described in \cref{sec:background_activation}.
|
||||||
|
|
||||||
Microcarriers have historically been used to grow a number of adherent cell
|
Microcarriers have historically been used to grow a number of adherent cell
|
||||||
types for a variety of applications. They were introduced in 1967 as a means to
|
types for a variety of applications. They were introduced in 1967 as a means to
|
||||||
grow adherent cells `in suspension', effectively turning a 2D flask system into
|
grow adherent cells ``in suspension,'' effectively turning a 2D flask into a 3D
|
||||||
a 3D culture system\cite{WEZEL1967}. Microcarriers are generally spherical and
|
culture system\cite{WEZEL1967}. Microcarriers are generally spherical and are
|
||||||
are several hundred \si{\um} in diameter, which means they collectively have a
|
several hundred \si{\um} in diameter, which means they collectively have a much
|
||||||
much higher surface area than a traditional flask when matched for volume.
|
higher surface area than a traditional flask when matched for volume.
|
||||||
Consequently, this means that microcarrier-based cultures can operate with much
|
Consequently, this means that microcarrier-based cultures can operate with much
|
||||||
lower footprints than flask-like systems. Microcarriers also allow cell culture
|
lower footprints than flask-like systems. Microcarriers also allow cell cultures
|
||||||
to operate more like a traditional chemical engineering process, wherein a
|
to operate more like traditional chemical engineering processes, wherein a
|
||||||
\gls{cstr} may be employed to enhance oxygen transfer, maintain pH,
|
\gls{cstr} may be employed to enhance oxygen transfer, maintain pH, and
|
||||||
and continuously supply nutrients\cite{Derakhti2019}.
|
continuously supply nutrients\cite{Derakhti2019}.
|
||||||
|
|
||||||
A variety of microcarriers have been designed, primarily differing in their
|
A variety of microcarriers have been designed, primarily differing in their
|
||||||
choice of material and macroporous structure. Key concerns driving these
|
choice of material and macroporous structure. Key concerns driving these choices
|
||||||
choiceshave been cell attachment at the beginning of culture and cell detachment
|
have been cell attachment at the beginning of culture and cell detachment at the
|
||||||
at the harvesting step\cite{Derakhti2019}. Many microcarriers simply use
|
harvesting step\cite{Derakhti2019}. Many microcarriers simply use polystyrene
|
||||||
polystyrene (the material used for tissue culture flasks and dishes in general)
|
(the material used for tissue culture flasks and dishes in general) with no
|
||||||
with no modification (SoloHill Plastic, Nunc Biosilon), with a cationic surface
|
modification (SoloHill Plastic, Nunc Biosilon), with a cationic surface charge
|
||||||
charge (SoloHill Hillex) or coated with an \gls{ecm} protein such as collagen
|
(SoloHill Hillex) or coated with an \gls{ecm} protein such as collagen (SoloHill
|
||||||
(SoloHill Fact III). Other base materials have been used such as dextran (GE
|
Fact III). Other base materials have been used such as dextran (GE Healthcare
|
||||||
Healthcare Cytodex), cellulose (GE Healthcare Cytopore), and glass (\sigald{}
|
Cytodex), cellulose (GE Healthcare Cytopore), and glass (\sigald{} G2767), all
|
||||||
G2767), all with none or similar surface modifications. Additionally, some
|
with similar surface modifications (if any). Additionally, some microcarriers
|
||||||
microcarriers such as \gls{cus} and \gls{cug} are composed entirely out of
|
such as \gls{cus} and \gls{cug} are composed entirely out of protein (in these
|
||||||
protein (in these cases, porcine collagen) which also allows the microcarriers
|
cases, porcine collagen) which also allows the microcarriers to be enzymatically
|
||||||
to be enzymatically degraded. In the case of non-protein materials, cells may
|
degraded. In the case of non-protein materials, cells may still be detached
|
||||||
still be detached using enzymes but these require similar methods to those
|
using enzymes but these require similar methods to those currently used in
|
||||||
currently used in flasks such as trypsin which target the cellular \gls{ecm}
|
flasks such as trypsin which target the cellular \gls{ecm} directly. Since
|
||||||
directly. Since trypsin and related enzymes tends to be harsh on cells, an
|
trypsin and related enzymes tends to be harsh on cells, an advantage of using
|
||||||
advantage of using entirely protein-based microcarriers is that they can be
|
entirely protein-based microcarriers is that they can be degraded using a much
|
||||||
degraded using a much safer enzyme such as collagenase, at the cost of being
|
safer enzyme such as collagenase, at the cost of being more expensive and also
|
||||||
more expensive and also being harder to make
|
being harder to make \gls{gmp}-compliant\cite{Derakhti2019}. Going one step
|
||||||
\gls{gmp}-compliant\cite{Derakhti2019}. Going one step further, some
|
further, some microcarriers are composed of a naturally degrading scaffold such
|
||||||
microcarriers are composed of a naturally degrading scaffold such as alginate,
|
as alginate, which do not need an enzyme for degradation. Finally, microcarriers
|
||||||
which do not need an enzyme for degradation. Finally, microcarriers can differ
|
can differ in their overall structure. \gls{cug} and \gls{cus} microcarriers as
|
||||||
in their overall structure. \gls{cug} and \gls{cus} microcarriers as well as the
|
well as the Cytopore microcarriers are macroporous, meaning they have a porous
|
||||||
Cytopore microcarriers are macroporous, meaning they have a porous network in
|
network in which cells can attach throughout their interior. This drastically
|
||||||
which cells can attach throughout their interior. This drastically increases the
|
increases the effective surface area and consequently the number of cells which
|
||||||
effective surface area and consequently the number of cells which may be grown
|
may be grown per unit volume. Other microcarriers are microporous (eg only
|
||||||
per unit volume. Other microcarriers are microporous (eg only to small
|
permeable to small molecules) or not porous at all; in either case, cells can
|
||||||
molecules) or not porous at all (eg polystyrene); in either case the cells can
|
only grow on the outer surface.
|
||||||
only grow on the surface.
|
|
||||||
|
|
||||||
Microcarriers in general have seen the most use in growing \gls{cho} cells and
|
Microcarriers have been mainly used for growing \gls{cho} cells and hybridomas
|
||||||
hybridomas in the case of protein manufacturing (eg \gls{igg}
|
in the case of protein manufacturing (eg \gls{igg} production)\cite{Xiao1999,
|
||||||
production)\cite{Xiao1999, Kim2011} as well as \glspl{esc} and \glspl{msc} more
|
Kim2011} as well as \glspl{esc} and \glspl{msc} more recently in the case of
|
||||||
recently in the case of cell manufacturing\cite{Heathman2015, Sart2011,
|
cell manufacturing\cite{Heathman2015, Sart2011, Chen2013, Schop2010, Rafiq2016}.
|
||||||
Chen2013, Schop2010, Rafiq2016}. Interestingly, some groups have even explored
|
Interestingly, some groups have even explored using biodegradable microcarriers
|
||||||
using biodegradable microcarriers \invivo{} as a delivery vehicle for stem cell
|
\invivo{} as a delivery vehicle for stem cell therapies in the context of
|
||||||
therapies in the context of regenerative medicine\cite{Zhang2016, Saltz2016,
|
regenerative medicine\cite{Zhang2016, Saltz2016, Park2013, Malda2006}. However,
|
||||||
Park2013, Malda2006}. However, the characteristic shared by all the cell types
|
all these cell types are adherent. In this work, we explore the use of
|
||||||
in this application is the fact that they are adherent. In this work, we explore
|
microcarrier for T cells, which are naturally non-adherent.
|
||||||
the use of microcarrier for T cells, which are naturally non-adherent.
|
|
||||||
|
|
||||||
The microcarriers used in this work were \gls{cus} and \gls{cug} (mostly the
|
The microcarriers used in this work were \gls{cus} and \gls{cug} (mostly the
|
||||||
former) which are both composed of cross-linked gelatin and have a macroporous
|
former) which are both composed of cross-linked gelatin and have a macroporous
|
||||||
|
@ -1119,7 +1114,7 @@ T cells naturally expand in the lymph nodes which have an \gls{ecm} composed of
|
||||||
collagen\cite{Dustin2001, Ebnet1996, Ohtani2008}. Despite this, T cells don't
|
collagen\cite{Dustin2001, Ebnet1996, Ohtani2008}. Despite this, T cells don't
|
||||||
interact with collagen fibers in the lymph node as the collagen fibers are
|
interact with collagen fibers in the lymph node as the collagen fibers are
|
||||||
sheathed with stromal fibroblasts\cite{Dustin2001, Ebnet1996}. However, the
|
sheathed with stromal fibroblasts\cite{Dustin2001, Ebnet1996}. However, the
|
||||||
\gls{ecm} of peripheral tissues is dense with exposed collagen fibers are
|
\gls{ecm} of peripheral tissues is dense where exposed collagen fibers are
|
||||||
available to interact with T cells. Furthermore, T cells have been shown
|
available to interact with T cells. Furthermore, T cells have been shown
|
||||||
\invitro{} to crawl along collagen fibers in the presence of \glspl{apc},
|
\invitro{} to crawl along collagen fibers in the presence of \glspl{apc},
|
||||||
facilitating short encounters with \glspl{apc}\cite{Gunzer2000}. While this may
|
facilitating short encounters with \glspl{apc}\cite{Gunzer2000}. While this may
|
||||||
|
@ -1129,16 +1124,15 @@ not be ideal \invivo{} due to the lack of cumulative signal received by
|
||||||
|
|
||||||
The major surface receptors for collagen are \gls{a2b1} and
|
The major surface receptors for collagen are \gls{a2b1} and
|
||||||
\gls{a2b2}\cite{Dustin2001, Hemler1990}. These receptors are not expressed on
|
\gls{a2b2}\cite{Dustin2001, Hemler1990}. These receptors are not expressed on
|
||||||
naive T cells and thus presence and stimulation of collagen alone is not
|
naive \gls{tn} cells and thus presence and stimulation of collagen alone is not
|
||||||
sufficient to cause activation or expansion of T cells\cite{Hemler1990}. These
|
sufficient for activation or expansion\cite{Hemler1990}; however, they have been
|
||||||
receptors have been shown to lead to a number of functions that may be useful in
|
shown to possess many functions that may be useful for T cell expansion. First,
|
||||||
the context of T cell expansion. First, they have been shown to act in a
|
they can act in a costimulatory manner which leads to increased
|
||||||
costimulatory manner which leads to increased proliferation\cite{Rao2000}.
|
proliferation\cite{Rao2000}. Furthermore, \gls{a2b1} and \gls{a2b2} seem to
|
||||||
Furthermore, \gls{a2b1} and \gls{a2b2} have been shown to protect Jurkat cells
|
protect Jurkat cells against Fas-mediated apoptosis in the presence of collagen
|
||||||
against Fas-mediated apoptosis in the presence of collagen I\cite{Aoudjit2000,
|
I\cite{Aoudjit2000, Gendron2003}. Finally, these receptors can increase
|
||||||
Gendron2003}. Finally, these receptors have been shown to increase \gls{ifng}
|
\gls{ifng} production \invitro{} when T cells derived from human \glspl{pbmc}
|
||||||
production \invitro{} when T cells derived from human \glspl{pbmc} are
|
are stimulated in the presence of collagen I\cite{Boisvert2007}.
|
||||||
stimulated in the presence of collagen I\cite{Boisvert2007}.
|
|
||||||
|
|
||||||
\subsection{The Role of IL15 in Memory T Cell Proliferation}
|
\subsection{The Role of IL15 in Memory T Cell Proliferation}
|
||||||
|
|
||||||
|
@ -1148,8 +1142,8 @@ further exploration in \cref{aim2b}.
|
||||||
|
|
||||||
Functionally, mice lacking the gene for either \il{15}\cite{Kennedy2000} or its
|
Functionally, mice lacking the gene for either \il{15}\cite{Kennedy2000} or its
|
||||||
high affinity receptor \il{15R$\upalpha$}\cite{Lodolce1998} are generally
|
high affinity receptor \il{15R$\upalpha$}\cite{Lodolce1998} are generally
|
||||||
healthy but show a deficit in memory CD8 T cells, thus underscoring its
|
healthy but show a deficit in memory CD8 T cells, thus underscoring this
|
||||||
importance in manufacturing high-quality memory T cells for immunotherapies. T
|
cytokine's importance in producing memory T cells for immunotherapies. T
|
||||||
cells themselves express \il{15} and all of its receptor
|
cells themselves express \il{15} and all of its receptor
|
||||||
components\cite{MirandaCarus2005}. Additionally, blocking \il{15} itself or
|
components\cite{MirandaCarus2005}. Additionally, blocking \il{15} itself or
|
||||||
\il{15R$\upalpha$} \invitro{} has been shown to inhibit homeostatic
|
\il{15R$\upalpha$} \invitro{} has been shown to inhibit homeostatic
|
||||||
|
@ -1158,20 +1152,20 @@ proliferation of resting human T cells\cite{MirandaCarus2005}.
|
||||||
% ACRO fix the il2R and IL15R stuff
|
% ACRO fix the il2R and IL15R stuff
|
||||||
\il{15} has been puzzling historically because it shares almost the same pathway
|
\il{15} has been puzzling historically because it shares almost the same pathway
|
||||||
as \il{2} yet has different functions\cite{Stonier2010, Osinalde2014, Giri1994,
|
as \il{2} yet has different functions\cite{Stonier2010, Osinalde2014, Giri1994,
|
||||||
Giri1995}. In particular, both cytokines share the common $\upgamma$ subchain
|
Giri1995}. In particular, both cytokines bond with heterotrimeric receptors
|
||||||
(CD132) as well as the \il{2} $\upbeta$ receptor (CD122). The main difference in
|
which share the common $\upgamma$ subchain (CD132) as well as the \il{2}
|
||||||
the heterodimeric receptors for \il{2} and \il{15} is the \il{2} $\upalpha$
|
$\upbeta$ receptor (CD122). The difference is the third subchain which is either
|
||||||
receptor (CD25) and the \il{15} $\upalpha$ chain respectively, both of which
|
the \il{2} $\upalpha$ receptor (CD25) or the \il{15} $\upalpha$ chain
|
||||||
have high affinity for their respective ligands. The \il{2R$\upalpha$} chain
|
respectively, both of which have high affinity for their respective ligands. The
|
||||||
itself does not have any signaling capacity, and therefore all the signaling
|
\il{2R$\upalpha$} chain itself does not have any signaling capacity, and
|
||||||
resulting from \il{2} is mediated thought the $\upbeta$ and $\upgamma$ chains,
|
therefore all the signaling resulting from \il{2} is mediated thought the
|
||||||
namely via JAK1 and JAK3 leading to STAT5 activation consequently T cell
|
$\upbeta$ and $\upgamma$ chains (namely via JAK1 and JAK3, which leads to STAT5
|
||||||
activation. \il{15R$\upalpha$} itself has some innate signaling capacity, but
|
activation, which leads to T cell activation). \il{15R$\upalpha$} itself has
|
||||||
this is poorly characterized in lymphocytes\cite{Stonier2010}. Thus there is a
|
some innate signaling capacity, but this is poorly characterized in
|
||||||
significant overlap between the functions of \il{2} and \il{15} due to their
|
lymphocytes\cite{Stonier2010}. Thus there is a significant overlap between the
|
||||||
receptors sharing the $\upbeta$ and $\upgamma$ chains in their heterodimeric
|
functions of \il{2} and \il{15} due to their receptors sharing the $\upbeta$ and
|
||||||
receptors, and perhaps the main driver of their differential functions it the
|
$\upgamma$ chains, and perhaps the main driver of their differential functions
|
||||||
half life of each respective receptor\cite{Osinalde2014}.
|
it the half life of each respective receptor\cite{Osinalde2014}.
|
||||||
|
|
||||||
Where \il{15} is unique is that many (or possibly most) of its functions derive
|
Where \il{15} is unique is that many (or possibly most) of its functions derive
|
||||||
from being membrane-bound to its receptor\cite{Stonier2010}. Particularly,
|
from being membrane-bound to its receptor\cite{Stonier2010}. Particularly,
|
||||||
|
@ -1186,18 +1180,18 @@ mechanism is that cells expression \il{15R$\upalpha$} either need to express
|
||||||
proximity require the $\upbeta$ and $\upgamma$ chains to receive the signal. In
|
proximity require the $\upbeta$ and $\upgamma$ chains to receive the signal. In
|
||||||
addition to \textit{trans} presentation, \il{15} may also work in a \textit{cis}
|
addition to \textit{trans} presentation, \il{15} may also work in a \textit{cis}
|
||||||
manner, where \il{15R$\upalpha$}/\il{15} complexes may bind to the $\upbeta$ and
|
manner, where \il{15R$\upalpha$}/\il{15} complexes may bind to the $\upbeta$ and
|
||||||
$\upgamma$ chains on the same cell, assuming all receptors are expressed and
|
$\upgamma$ chains on the same cell, assuming each subchain is expressed and
|
||||||
soluble \il{15} is available\cite{Olsen2007}. Finally, \il{15R$\upalpha$} itself can exist in
|
soluble \il{15} is available\cite{Olsen2007}. Finally, \il{15R$\upalpha$} itself
|
||||||
a soluble form, which can bind to \il{15} and signal to cells which are not
|
can exist in soluble form, which can bind to \il{15} and signal to cells which
|
||||||
adjacent to the source independent of the \textit{cis/trans} mechanisms already
|
are not adjacent to the source independent of the \textit{cis/trans} mechanisms
|
||||||
described\cite{Budagian2004}.
|
already described\cite{Budagian2004}.
|
||||||
|
|
||||||
\subsection{Overview of Design of Experiments}\label{sec:background_doe}
|
\subsection{Overview of Design of Experiments}\label{sec:background_doe}
|
||||||
|
|
||||||
The \gls{dms} system described in this dissertation has a number of parameters
|
The \gls{dms} system described in this dissertation has many parameters that can
|
||||||
that can be optimized and controlled (eg \glspl{cpp}). A \gls{doe} is an ideal
|
be optimized and controlled (eg \glspl{cpp}). A \gls{doe} is an ideal framework
|
||||||
framework to test multiple parameters simultaneously and determine which are
|
to test multiple parameters simultaneously and determine which are relevant
|
||||||
relevant \glspl{cpp}.
|
\glspl{cpp}.
|
||||||
|
|
||||||
The goal of \gls{doe} is to answer a data-driven question with the least number
|
The goal of \gls{doe} is to answer a data-driven question with the least number
|
||||||
of resources\cite{Wu2009}. It was developed in many non-biological industries
|
of resources\cite{Wu2009}. It was developed in many non-biological industries
|
||||||
|
@ -1206,30 +1200,33 @@ industries where engineers needed to minimize downtime and resource consumption
|
||||||
on full-scale production lines.
|
on full-scale production lines.
|
||||||
|
|
||||||
At its core, a \gls{doe} is simply a matrix of conditions to test where each row
|
At its core, a \gls{doe} is simply a matrix of conditions to test where each row
|
||||||
(usually called a `run') corresponds to one experimental unit for which the
|
(usually called a ``run,'' which is the term used throughout this work)
|
||||||
conditions are applied, and each column represents a parameter of concern to be
|
corresponds to one experimental unit for which the conditions are applied, and
|
||||||
tested. The values in each cell represent the level at which each parameter is
|
each column represents a parameter of concern to be tested. The values in each
|
||||||
to be tested. When the experiment is performed using this matrix of conditions,
|
cell represent the level of each parameter. When the experiment is performed
|
||||||
the results are be summarized into one or more `responses' that correspond to
|
using this matrix of conditions, the results are be summarized into one or more
|
||||||
each run. These responses are then be modeled (usually using linear regression)
|
``responses'' that correspond to each run. These responses are then be modeled
|
||||||
to determine the statistic relationship (also called an `effect') between each
|
(usually using linear regression) to determine the statistical relationship
|
||||||
parameter and the response(s).
|
(also called an ``effect'') between each parameter and the response(s).
|
||||||
|
|
||||||
Collectively, the space spanned by all parameters at their feasible ranges is
|
Collectively, the space spanned by all parameters at their feasible ranges is
|
||||||
commonly referred to as the `design space', and generally the goal of a
|
commonly referred to as the ``design space'', and generally the goal of a
|
||||||
\gls{doe} is to explore this design space using using the least number of runs
|
\gls{doe} is to explore this design space using using the least number of runs
|
||||||
possible. While there are many types of \glspl{doe} depending on the nature
|
possible. While there are many types of \glspl{doe} depending on the nature
|
||||||
of the parameters and the goal of the experimenter, they all share common
|
of the parameters and the goal of the experimenter, they all share common
|
||||||
principles:
|
principles:
|
||||||
|
|
||||||
\begin{description}
|
\begin{description}
|
||||||
\item [randomization --] The order in which the runs are performed should
|
\item [randomization --] The order in which the runs are performed should be
|
||||||
ideally be as random as possible. This is to mitigate against any confounding
|
randomized. This is to guarantee that the tested parameters are independent of
|
||||||
factors that may be present which depend on the order or position of the runs.
|
any unobserved influences to the response, and thus allows the causal effect
|
||||||
For an example in context, the evaporation rate of media in a tissue culture
|
of each parameter to be isolated completely\footnote{this is why \glspl{doe}
|
||||||
plate will be much faster at the perimeter of the plate vs the center. While
|
are sometimes called ``black box models;'' one can can safely say ``this
|
||||||
randomization does not eliminate this error, it will ensure the error is
|
parameter causes that'' without paying attention to the causal structure of
|
||||||
`spread' evenly across all runs in an unbiased manner.
|
the experiment}. For an example in context, the evaporation rate of media in
|
||||||
|
a tissue culture plate will be much faster at the perimeter of the plate vs
|
||||||
|
the center. While randomization does not eliminate this error, it will ensure
|
||||||
|
the error is ``spread'' across all runs in an unbiased manner.
|
||||||
\item [replication --] Since the analysis of a \gls{doe} is inherently
|
\item [replication --] Since the analysis of a \gls{doe} is inherently
|
||||||
statistical, replicates should be used to ensure that the underlying
|
statistical, replicates should be used to ensure that the underlying
|
||||||
distribution of errors can be estimated. While this is not strictly necessary
|
distribution of errors can be estimated. While this is not strictly necessary
|
||||||
|
@ -1237,7 +1234,7 @@ principles:
|
||||||
strong assumptions about the model structure (particularly in the case of
|
strong assumptions about the model structure (particularly in the case of
|
||||||
high-complexity models which could easily fit the data perfectly) and also
|
high-complexity models which could easily fit the data perfectly) and also
|
||||||
precludes the use of statistical tests such as the lack-of-fit test which can
|
precludes the use of statistical tests such as the lack-of-fit test which can
|
||||||
be useful in rejecting or accepting a particular analysis. Note that the
|
be useful in rejecting or accepting a particular model. Note that the
|
||||||
subject of replication is within but not the same as power analysis, which
|
subject of replication is within but not the same as power analysis, which
|
||||||
concerns the number of runs required to estimate a certain effect size.
|
concerns the number of runs required to estimate a certain effect size.
|
||||||
\item [orthogonality --] Orthogonality refers to the independence of each
|
\item [orthogonality --] Orthogonality refers to the independence of each
|
||||||
|
@ -1249,14 +1246,15 @@ principles:
|
||||||
experiments with many categorical variables) strategies exist to maximize
|
experiments with many categorical variables) strategies exist to maximize
|
||||||
orthogonality.
|
orthogonality.
|
||||||
\item [blocking --] In the case where the experiment must be non-randomly spread
|
\item [blocking --] In the case where the experiment must be non-randomly spread
|
||||||
over multiple groups, runs are assigned to `blocks' which are not necessarily
|
over multiple groups, runs are assigned to ``blocks'' which are not
|
||||||
relevant to the goals of the experiment but nonetheless could affect the
|
necessarily relevant to the goals of the experiment but nonetheless could
|
||||||
response. A key assumption that is (usually) made in the case of blocking is
|
affect the response. A key assumption that is (usually) made in the case of
|
||||||
that there is no interaction between the blocking variable and any of the
|
blocking is that there is no interaction between the blocking variable and any
|
||||||
experimental parameters. For example, in T cell expansion, if media lot were a
|
of the experimental parameters. For example, in T cell expansion, if media lot
|
||||||
blocking variable and expansion method were a parameter, we would by default
|
were a blocking variable and expansion method were a parameter, we would by
|
||||||
assume that the effect of the expansion method does not depend on the media
|
default assume that the effect of the expansion method does not depend on the
|
||||||
lot (even if the media lot itself might change the mean of the response).
|
media lot (even if the media lot itself might change the mean of the
|
||||||
|
response).
|
||||||
\end{description}
|
\end{description}
|
||||||
|
|
||||||
\Glspl{doe} served three purposes in this dissertation. First, we used them as
|
\Glspl{doe} served three purposes in this dissertation. First, we used them as
|
||||||
|
@ -1279,28 +1277,26 @@ investigated. However, it could be the case that one already has data on many of
|
||||||
the factors of concern. If one only cares about main effects, performing a
|
the factors of concern. If one only cares about main effects, performing a
|
||||||
\gls{doe} (particularly a lower-powered screening experiment such as a
|
\gls{doe} (particularly a lower-powered screening experiment such as a
|
||||||
resolution III design) with these factors and a few others may not be
|
resolution III design) with these factors and a few others may not be
|
||||||
productive, and one is better off performed a few extra pilot experiments before
|
productive, and one is better off performing a few extra pilot experiments
|
||||||
doing a more complex design such as a central composite if desired. Furthermore,
|
before doing a more complex design such as a central composite if desired.
|
||||||
it should be noted that while the goal of a \gls{doe} is to minimize resources,
|
Furthermore, it should be noted that while the goal of a \gls{doe} is to
|
||||||
the size necessary to justify a \gls{doe} may not be worth the experimental
|
minimize resources, the size necessary to justify a \gls{doe} may not be worth
|
||||||
return. For biological work (or any domain with little automation), performing a
|
the experimental return. For biological work (or any domain with little
|
||||||
randomized experiment with 20 to 30 runs is not trivial from a logistical
|
automation), performing a randomized experiment with 20 to 30 runs is not
|
||||||
perspective, especially when considering the number of manual manipulations and
|
trivial from a logistical perspective, especially when considering the number of
|
||||||
the chance of human error.
|
manual manipulations and the chance of human error.
|
||||||
|
|
||||||
Despite these caveats, many of the principles used for a \gls{doe} are important
|
Despite these caveats, many of the principles used for a \gls{doe} are important
|
||||||
in general for experimentation. The most obvious is randomization, which is
|
in general for experimentation. The most obvious is randomization, which is
|
||||||
often not employed (and also not explicitly mentioned in papers) even though it
|
often not employed (and also not explicitly mentioned in papers). Assuming the
|
||||||
is empirically obvious that well plates have different evaporation rates
|
experiment is manual, the largest reason to avoid randomization is that the
|
||||||
depending on well position. Assuming the experiment is manual, the largest
|
experimentalist has no pattern to follow when administering treatment (such as
|
||||||
reason to avoid randomization is that the experimentalist has no pattern to
|
``add X to row 1 in well plate''), thus cognitive burden and the likelihood of
|
||||||
follow when administering treatment (such as ``add X to row 1 in well plate''),
|
mistakes increases. While \glspl{doe} are usually bigger with more parameters,
|
||||||
thus cognitive burden and the likelihood of mistakes increases. While
|
the one-factor-at-a-time experiment usually performed in biological disciplines
|
||||||
\glspl{doe} are usually bigger with more parameters, the one-factor-at-a-time
|
is much smaller and only has a few parameters, thus these concerns are minimal.
|
||||||
experiment usually performed in biological disciplines is much smaller and only
|
There is no reason to avoid randomization in these cases, as the added cognitive
|
||||||
has a few parameters, thus these concerns are minimal. There is no reason to
|
cost is far offset by the guarantee of eliminated bias due to run position.
|
||||||
avoid randomization in these cases, as the added cognitive cost is far offset by
|
|
||||||
the guarantee of eliminated bias due to run position.
|
|
||||||
|
|
||||||
\subsection{Identification and Standardization of CPPs and
|
\subsection{Identification and Standardization of CPPs and
|
||||||
CQAs}\label{sec:background_cqa}
|
CQAs}\label{sec:background_cqa}
|
||||||
|
@ -1315,15 +1311,15 @@ secrete numerous cytokines and metabolites in the media, which may reflect the
|
||||||
internal state accurately and thus serve as a potential set of \glspl{cqa}.
|
internal state accurately and thus serve as a potential set of \glspl{cqa}.
|
||||||
|
|
||||||
The complexity of these pathways dictates that we take a big-data approach to
|
The complexity of these pathways dictates that we take a big-data approach to
|
||||||
this problem. To this end, there are several pertinent multi-omic (or simply
|
this problem. To this end, there are several multi-omic (or simply ``omic'')
|
||||||
`omic') techniques that can be used to collect such datasets, which can then be
|
techniques that can be used to collect such datasets, which can then be fit to
|
||||||
mined, modeled, and correlated to relevent responses (such as an endpoint
|
relevent responses (such as an endpoint quantification of memory T cells) to
|
||||||
quantification of memory T cells) to identify pertinent \glspl{cqa}.
|
identify pertinent \glspl{cqa}.
|
||||||
|
|
||||||
An overview of the techniques used in this work are:
|
An overview of the techniques used in this work are:
|
||||||
|
|
||||||
\begin{description}
|
\begin{description}
|
||||||
\item[Luminex --] This is a multiplexed bead-based assay similar to \gls{elisa} that can measure
|
\item[luminex --] This is a multiplexed bead-based assay similar to \gls{elisa} that can measure
|
||||||
many bulk (not single cell) cytokine concentrations simultaneously
|
many bulk (not single cell) cytokine concentrations simultaneously
|
||||||
in a media sample. This is a destructive assay but does not require cells to
|
in a media sample. This is a destructive assay but does not require cells to
|
||||||
obtain a measurement.
|
obtain a measurement.
|
||||||
|
@ -1333,7 +1329,7 @@ An overview of the techniques used in this work are:
|
||||||
oxidation\cite{Buck2016, van_der_Windt_2012}. \gls{nmr} is a technique that
|
oxidation\cite{Buck2016, van_der_Windt_2012}. \gls{nmr} is a technique that
|
||||||
can non-destructively quantify small molecules in a media sample, and thus is
|
can non-destructively quantify small molecules in a media sample, and thus is
|
||||||
an attractive method that could be used for inline, real-time monitoring.
|
an attractive method that could be used for inline, real-time monitoring.
|
||||||
\item[Flow and Mass Cytometry --] Flow cytometry using fluorophores has been
|
\item[flow and mass cytometry --] Flow cytometry using fluorophores has been
|
||||||
used extensively for immune cell analysis, but has a practical limit of
|
used extensively for immune cell analysis, but has a practical limit of
|
||||||
approximately 18 colors\cite{Spitzer2016}. Mass cytometry is analogous to
|
approximately 18 colors\cite{Spitzer2016}. Mass cytometry is analogous to
|
||||||
traditional flow cytometry except that it uses heavy-metal \gls{mab}
|
traditional flow cytometry except that it uses heavy-metal \gls{mab}
|
||||||
|
@ -1359,11 +1355,11 @@ interesting cell types and the markers that define them.
|
||||||
|
|
||||||
Ultimately, identifying \glspl{cqa} will likely be an iterative process, wherein
|
Ultimately, identifying \glspl{cqa} will likely be an iterative process, wherein
|
||||||
putative \glspl{cqa} will be identified, the corresponding \glspl{cpp} will be
|
putative \glspl{cqa} will be identified, the corresponding \glspl{cpp} will be
|
||||||
set and optimized to maximize products with these \glspl{cpp}, and then
|
set to maximize high-quality products, and then additional data will be
|
||||||
additional data will be collected in the clinic as the product is tested on
|
collected in the clinic as the product is tested on various patients with
|
||||||
various patients with different indications. Additional \glspl{cqa} may be
|
different indications. Additional \glspl{cqa} may be identified which better
|
||||||
identified which better predict specific clinical outcomes, which can be fed
|
predict specific clinical outcomes, which can be fed back into the process model
|
||||||
back into the process model and optimized again.
|
and optimized again.
|
||||||
|
|
||||||
\section{Innovation}
|
\section{Innovation}
|
||||||
|
|
||||||
|
@ -1373,7 +1369,7 @@ novel considering the state-of-the-art technology for T cell manufacturing:
|
||||||
\begin{itemize}
|
\begin{itemize}
|
||||||
\item \Glspl{dms} offers a compelling alternative to state-of-the-art magnetic
|
\item \Glspl{dms} offers a compelling alternative to state-of-the-art magnetic
|
||||||
bead technologies (e.g. DynaBeads, MACS-Beads), which is noteworthy because
|
bead technologies (e.g. DynaBeads, MACS-Beads), which is noteworthy because
|
||||||
the licenses for these techniques is controlled by only a few companies
|
the licenses for these techniques are controlled by only a few companies
|
||||||
(Invitrogen and Miltenyi respectively). Because of this, bead-based expansion
|
(Invitrogen and Miltenyi respectively). Because of this, bead-based expansion
|
||||||
is more expensive to implement and therefore hinders companies from entering
|
is more expensive to implement and therefore hinders companies from entering
|
||||||
the rapidly growing T cell manufacturing arena. Providing an alternative will
|
the rapidly growing T cell manufacturing arena. Providing an alternative will
|
||||||
|
@ -1381,7 +1377,7 @@ novel considering the state-of-the-art technology for T cell manufacturing:
|
||||||
lower costs, and higher innovation in the T cell manufacturing space.
|
lower costs, and higher innovation in the T cell manufacturing space.
|
||||||
\item This is the first technology for T cell immunotherapies that selectively
|
\item This is the first technology for T cell immunotherapies that selectively
|
||||||
expands memory T cell populations with greater efficiency relative to
|
expands memory T cell populations with greater efficiency relative to
|
||||||
bead-based expansion Others have demonstrated methods that can achieve greater
|
bead-based expansion. Others have demonstrated methods that can achieve greater
|
||||||
expansion of T cells, but not necessarily specific populations that are known
|
expansion of T cells, but not necessarily specific populations that are known
|
||||||
to be potent.
|
to be potent.
|
||||||
\item We used \glspl{doe} to discover and validate novel \glspl{cpp}, which is a
|
\item We used \glspl{doe} to discover and validate novel \glspl{cpp}, which is a
|
||||||
|
@ -1389,9 +1385,9 @@ novel considering the state-of-the-art technology for T cell manufacturing:
|
||||||
usage in the development of cell therapies where research often employs a
|
usage in the development of cell therapies where research often employs a
|
||||||
one-factor-at-a-time approach. We believe this method is highly relevant to
|
one-factor-at-a-time approach. We believe this method is highly relevant to
|
||||||
the development of cell therapies, not only for process optimization but also
|
the development of cell therapies, not only for process optimization but also
|
||||||
hypotheses generation. Furthermore, it is a perfectly natural strategy to use
|
hypotheses generation. Furthermore, it is a natural strategy to use even at
|
||||||
even at small scale, where the cost of reagents, cells, and materials often
|
small scale, where the cost of reagents, cells, and materials often precludes
|
||||||
precludes large sample sizes.
|
large sample sizes.
|
||||||
\item The \gls{dms} system is be compatible with static bioreactors such as the
|
\item The \gls{dms} system is be compatible with static bioreactors such as the
|
||||||
G-Rex which has been adopted throughout the cell therapy industry. Thus this
|
G-Rex which has been adopted throughout the cell therapy industry. Thus this
|
||||||
technology can be easily incorporated into existing cell therapy process that
|
technology can be easily incorporated into existing cell therapy process that
|
||||||
|
|
Loading…
Reference in New Issue