diff --git a/tex/references.bib b/tex/references.bib index b02360e..847de78 100644 --- a/tex/references.bib +++ b/tex/references.bib @@ -2596,6 +2596,57 @@ CONCLUSIONS: We developed a simplified, semi-closed system for the initial selec publisher = {Springer Science and Business Media {LLC}}, } +@Article{Guerra2001, + author = {S. Del Guerra and C. Bracci and K. Nilsson and A. Belcourt and L. Kessler and R. Lupi and L. Marselli and P. De Vos and P. Marchetti}, + journal = {Biotechnology and Bioengineering}, + title = {Entrapment of dispersed pancreatic islet cells in {CultiSpher}-S macroporous gelatin microcarriers: Preparation, in vitro characterization, and microencapsulation}, + year = {2001}, + number = {6}, + pages = {741--744}, + volume = {75}, + doi = {10.1002/bit.10053}, + publisher = {Wiley}, +} + +@Article{Fernandes2007, + author = {A.M. Fernandes and T.G. Fernandes and M.M. Diogo and C. Lobato da Silva and D. Henrique and J.M.S. Cabral}, + journal = {Journal of Biotechnology}, + title = {Mouse embryonic stem cell expansion in a microcarrier-based stirred culture system}, + year = {2007}, + month = {oct}, + number = {2}, + pages = {227--236}, + volume = {132}, + doi = {10.1016/j.jbiotec.2007.05.031}, + publisher = {Elsevier {BV}}, +} + +@Article{Storm_2010, + author = {Michael P. Storm and Craig B. Orchard and Heather K. Bone and Julian B. Chaudhuri and Melanie J. Welham}, + journal = {Biotechnology and Bioengineering}, + title = {Three-dimensional culture systems for the expansion of pluripotent embryonic stem cells}, + year = {2010}, + month = {jun}, + number = {4}, + pages = {683--695}, + volume = {107}, + doi = {10.1002/bit.22850}, + publisher = {Wiley}, +} + +@Article{Eibes2010, + author = {Gemma Eibes and Francisco dos Santos and Pedro Z. Andrade and Joana S. Boura and Manuel M.A. Abecasis and Cl{\'{a}}udia Lobato da Silva and Joaquim M.S. Cabral}, + journal = {Journal of Biotechnology}, + title = {Maximizing the ex vivo expansion of human mesenchymal stem cells using a microcarrier-based stirred culture system}, + year = {2010}, + month = {apr}, + number = {4}, + pages = {194--197}, + volume = {146}, + doi = {10.1016/j.jbiotec.2010.02.015}, + publisher = {Elsevier {BV}}, +} + @Comment{jabref-meta: databaseType:bibtex;} @Comment{jabref-meta: grouping: diff --git a/tex/thesis.tex b/tex/thesis.tex index 50562c7..c41e098 100644 --- a/tex/thesis.tex +++ b/tex/thesis.tex @@ -1,4 +1,3 @@ -% \documentclass[twocolumn]{article} \documentclass{report} \usepackage[section]{placeins} \usepackage[top=1in,left=1.5in,right=1in,bottom=1in]{geometry} @@ -189,6 +188,8 @@ \newacronym{aws}{AWS}{amazon web services} \newacronym{qpcr}{qPCR}{quantitative polymerase chain reaction} \newacronym{cstr}{CSTR}{continuously stirred tank bioreactor} +\newacronym{esc}{ESC}{embryonic stem cell} +\newacronym{msc}{MSC}{mesenchymal stromal cells} %%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%% % SI units for uber nerds @@ -455,9 +456,6 @@ quality in an industrial setting. \section*{overview} -% TODO this is basically the same as the first part of the backgound, I guess I -% can just trim it down - T cell-based immunotherapies have received great interest from clinicians and industry due to their potential to treat, and often cure, cancer and other diseases\cite{Fesnak2016,Rosenberg2015}. In 2017, Novartis and Kite Pharma @@ -465,86 +463,63 @@ received FDA approval for \textit{Kymriah} and \textit{Yescarta} respectively, two genetically-modified \gls{car} T cell therapies against B cell malignancies. Despite these successes, \gls{car} T cell therapies are constrained by an expensive and difficult-to-scale manufacturing process with little control on -cell quality and phenotype3,4. State-of-the-art T cell manufacturing techniques -focus on \acd{3} and \acd{28} activation and expansion, typically -presented on superparamagnetic, iron-based microbeads (Invitrogen Dynabead, -Miltenyi MACS beads), on nanobeads (Miltenyi TransACT), or in soluble tetramers -(Expamer)\cite{Roddie2019,Dwarshuis2017,Wang2016, Piscopo2017, Bashour2015}. -These strategies overlook many of the signaling components present in the -secondary lymphoid organs where T cells expand \invivo{}. Typically, T cells are -activated under close cell-cell contact, which allows for efficient -autocrine/paracrine signaling via growth-stimulating cytokines such as -\gls{il2}. Additionally, the lymphoid tissues are comprised of \gls{ecm} -components such as collagen, which provide signals to upregulate proliferation, -cytokine production, and pro-survival pathways\cite{Gendron2003, Ohtani2008, - Boisvert2007, Ben-Horin2004}. We hypothesized that culture conditions that -better mimic these \invivo{} expansion conditions of T cells, can significantly -improve the quality and quantity of manufactured T cells and provide better -control on the resulting T cell phenotype. +cell quality and phenotype\cite{Roddie2019, Dwarshuis2017}. State-of-the-art T +cell manufacturing techniques focus on \acd{3} and \acd{28} activation and +expansion, typically presented on superparamagnetic, iron-based microbeads +(Invitrogen Dynabead, Miltenyi MACS beads), on nanobeads (Miltenyi TransACT), or +in soluble tetramers (Expamer)\cite{Roddie2019,Dwarshuis2017,Wang2016, + Piscopo2017, Bashour2015}. These strategies overlook many of the signaling +components present in the secondary lymphoid organs where T cells expand +\invivo{}. Typically, T cells are activated under close cell-cell contact, which +allows for efficient autocrine/paracrine signaling via growth-stimulating +cytokines such as \gls{il2}. Additionally, the lymphoid tissues are comprised of +\gls{ecm} components such as collagen and stromal cells, which provide signals +to upregulate proliferation, cytokine production, and pro-survival +pathways\cite{Gendron2003, Ohtani2008, Boisvert2007, Ben-Horin2004}. A variety of solutions have been proposed to make the T cell expansion process -more physiological. One strategy is to use modified feeder cell cultures to -provide activation signals similar to those of \glspl{dc}\cite{Forget2014}. -While this has the theoretical capacity to mimic many components of the lymph -node, it is hard to reproduce on a large scale due to the complexity and -inherent variability of using cell lines in a fully \gls{gmp}-compliant manner. -Others have proposed biomaterials-based solutions to circumvent this problem, -including lipid-coated microrods\cite{Cheung2018}, 3D-scaffolds via either -Matrigel\cite{Rio2018} or 3d-printed lattices\cite{Delalat2017}, ellipsoid -beads\cite{meyer15_immun}, and \gls{mab}-conjugated \gls{pdms} -beads\cite{Lambert2017} that respectively recapitulate the cellular membrane, -large interfacial contact area, 3D-structure, or soft surfaces T cells normally -experience \invivo{}. While these have been shown to provide superior expansion -compared to traditional microbeads, none of these methods has been able to show -preferential expansion of functional naïve/memory and CD4 T cell populations. -Generally, T cells with a lower differentiation state such as naïve and memory -cells have been shown to provide superior anti-tumor potency, presumably due to -their higher potential to replicate, migrate, and engraft, leading to a -long-term, durable response\cite{Xu2014, Fraietta2018, Gattinoni2011, - Gattinoni2012}. Likewise, CD4 T cells are similarly important to anti-tumor -potency due to their cytokine release properties and ability to resist -exhaustion\cite{Wang2018, Yang2017}. Therefore, methods to increase naïve/memory -and CD4 T cells in the final product are needed, a critical consideration being -ease of translation to industry and ability to interface with scalable systems -such as bioreactors. +more physiological. Including feeder cell cultures\cite{Forget2014} and +biomaterials-based methods such as lipid-coated microrods or 3D scaffold +gels\cite{Cheung2018,Delalat2017,meyer15_immun,Lambert2017} that attempt to +recapitulate the cellular membrane, large interfacial contact area, +3D-structure, or soft surfaces T cells normally experience \invivo{}. While +these have been shown to activation and expand T cells, they either are not +scalable (in the case of feeder cells) or still lack many of the signals and +cues T cells experience as the expand. Additionally, none have been shown to +preferentially expand highly-potent T cell necessary for anti-cancer therapies. +Such high potency cells including subtypes with low differentiation state such +as \gls{tscm} and \gls{tcm} cells or CD4 cells, all of which have been shown to +be necessary for durable responses\cite{Xu2014, Fraietta2018, Gattinoni2011, + Gattinoni2012,Wang2018, Yang2017}. Methods to increase memory and CD4 T cells +in the final product are needed. Furthermore, \gls{qbd} principles such as +discovering and validating novel \glspl{cqa} and \glspl{cpp} in the space of T +cell manufacturing are required to reproducibly manufacture these subtypes and +ensure low-cost and safe products with maximal effectiveness in the clinic -% TODO probably need to address some of the modeling stuff here as well - -This thesis describes a novel degradable microscaffold-based method derived from +This dissertation describes a novel \acrlong{dms}-based method derived from porous microcarriers functionalized with \acd{3} and \acd{28} \glspl{mab} for use in T cell expansion cultures. Microcarriers have historically been used -throughout the bioprocess industry for adherent cultures such as stem cells and -\gls{cho} cells, but not with suspension cells such as T -cells\cite{Heathman2015, Sart2011}. The microcarriers chosen to make the DMSs in -this study have a microporous structure that allows T cells to grow inside and -along the surface, providing ample cell-cell contact for enhanced autocrine and -paracrine signaling. Furthermore, the carriers are composed of gelatin, which is -a collagen derivative and therefore has adhesion domains that are also present -within the lymph nodes. Finally, the 3D surface of the carriers provides a -larger contact area for T cells to interact with the \glspl{mab} relative to -beads; this may better emulate the large contact surface area that occurs -between T cells and \glspl{dc}. These microcarriers are readily available in -over 30 countries and are used in an FDA fast-track-approved combination retinal -pigment epithelial cell product (Spheramine, Titan -Pharmaceuticals)\cite{purcellmain}. This regulatory history will aid in clinical -translation. We show that compared to traditional microbeads, \gls{dms}-expanded -T cells not only provide superior expansion, but consistently provide a higher -frequency of naïve/memory and CD4 T cells (CCR7+CD62L+) across multiple donors. -We also demonstrate functional cytotoxicity using a CD19 \gls{car} and a -superior performance, even at a lower \gls{car} T cell dose, of -\gls{dms}-expanded \gls{car}-T cells \invivo{} in a mouse xenograft model of -human B cell \gls{all}. Our results indicate that \glspl{dms} provide a robust -and scalable platform for manufacturing therapeutic T cells with higher -naïve/memory phenotype and more balanced CD4+ T cell content. +throughout the bioprocess industry for adherent cultures such as \gls{cho} cells +but not with suspension cells such as T cells\cite{Heathman2015, Sart2011}. The +microcarriers chosen to make the \gls{dms} in this work have a microporous +structure that allows T cells to grow inside and along the surface, providing +ample cell-cell contact for enhanced autocrine and paracrine signaling. +Furthermore, the 3D surface of the carriers provides a larger contact area for T +cells to interact with the \glspl{mab} relative to beads; this may better +emulate the large contact surface area that occurs between T cells and +\glspl{dc}. \section*{hypothesis} The hypothesis of this dissertation was that using \glspl{dms} created from off-the-shelf microcarriers and coated with activating \glspl{mab} would lead to higher quantity and quality T cells as compared to state-of-the-art bead-based -expansion. The objective of this dissertation was to develop this platform, test -its effectiveness both \invivo{} and \invivo{}, and develop computational -pipelines that could be used in a manufacturing environment. +expansion. We also hypothesized that T cells have measurable biological +signatures that are predictive of downstream outcomes and phenotypes. The +objective of this dissertation was to develop this platform, test its +effectiveness both \invitro{} and \invivo{}, and develop computational pipelines +to discover novel \glspl{cpp} and \glspl{cqa} that can be translated to a +manufacturing environment and a clinical trial setting. \section*{specific aims} @@ -575,7 +550,7 @@ used to generate functional \gls{car} T cells targeted toward CD19. \subsection*{aim 2: develop methods to control and predict T cell quality} For this second aim, we investigated methods to identify and control \glspl{cqa} -and glspl{cpp} for manufacturing T cells using the \gls{dms} platform. This was +and \glspl{cpp} for manufacturing T cells using the \gls{dms} platform. This was accomplished through two sub-aims: \begin{itemize} @@ -594,11 +569,12 @@ cells compared to state-of-the-art beads using \invivo{} mouse models for \section*{outline} -In Chapter~\ref{background}, we provide additional background on the current -state of T cell manufacturing and how the work in this dissertation moves the -field forward. In Chapters~\ref{aim1},~\ref{aim2a},~\ref{aim2b}, and~\ref{aim3} -we present the work pertaining to Aims 1, 2, and 3 respectively. Finally, we -present our final conclusions in Chapter~\ref{conclusions}. +In \cref{background}, we provide additional background on the current state of T +cell manufacturing and how the work in this dissertation moves the field +forward. In \cref{aim1,aim2a,aim2b,aim3} we present the work pertaining to Aims +1, 2a, 2b, and 3 respectively. Finally, in \cref{conclusions} we present our +conclusions as well as provide insights for how this work can be extended in the +future. \chapter{background and significance}\label{background} \section*{background} @@ -793,9 +769,9 @@ per unit volume. Other microcarriers are microporous (eg only to small molecules) or not porous at all (eg polystyrene) in which case the cells can only grow on the surface. -Microcarriers have seen the most use in growing \gls{cho} cells and hybridomas -in the case of protein manufacturing (eg \gls{igg} production)\cite{Xiao1999, - Kim2011} as well as pluripotent stem cells and mesenchymal stromal cells more +Microcarriers in general have seen the most use in growing \gls{cho} cells and +hybridomas in the case of protein manufacturing (eg \gls{igg} +production)\cite{Xiao1999, Kim2011} as well as \glspl{esc} and \glspl{msc} more recently in the case of cell manufacturing\cite{Heathman2015, Sart2011, Chen2013, Schop2010, Rafiq2016}. Interestingly, some groups have even explored using biodegradable microcarriers \invivo{} as a delivery vehicle for stem cell @@ -804,6 +780,15 @@ therapies in the context of regenerative medicine\cite{Zhang2016, Saltz2016, in this application is the fact that they are adherent. In this work, we explore 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 +former) which are both composed of cross-linked gelatin and have a macroporous +morphology. These specific carriers have been used in the past for pancreatic +islet cells\cite{Guerra2001}, \glspl{esc}\cite{Fernandes2007, Storm_2010}, and +\glspl{msc}\cite{Eibes2010}. Furthermore, they are readily available in over 30 +countries and are used in an FDA fast-track-approved combination retinal pigment +epithelial cell product (Spheramine, Titan Pharmaceuticals)\cite{purcellmain}. +This regulatory history will aid in clinical translation. + \subsection{methods to scale T cells} In order to scale T cell therapies to meet clinical demands, automation and