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\begin{thebibliography}{10}
\expandafter\ifx\csname url\endcsname\relax
\def\url#1{\texttt{#1}}\fi
\expandafter\ifx\csname urlprefix\endcsname\relax\def\urlprefix{URL }\fi
\providecommand{\bibinfo}[2]{#2}
\providecommand{\eprint}[2][]{\url{#2}}
\bibitem{Fesnak2016}
\bibinfo{author}{Fesnak, A.~D.}, \bibinfo{author}{June, C.~H.} \&
\bibinfo{author}{Levine, B.~L.}
\newblock \bibinfo{title}{Engineered t cells: the promise and challenges of
cancer immunotherapy}.
\newblock \emph{\bibinfo{journal}{Nature Reviews Cancer}}
\textbf{\bibinfo{volume}{16}}, \bibinfo{pages}{566--581}
(\bibinfo{year}{2016}).
\bibitem{Rosenberg2015}
\bibinfo{author}{Rosenberg, S.~A.} \& \bibinfo{author}{Restifo, N.~P.}
\newblock \bibinfo{title}{Adoptive cell transfer as personalized immunotherapy
for human cancer}.
\newblock \emph{\bibinfo{journal}{Science}} \textbf{\bibinfo{volume}{348}},
\bibinfo{pages}{62--68} (\bibinfo{year}{2015}).
\bibitem{Roddie2019}
\bibinfo{author}{Roddie, C.}, \bibinfo{author}{O'Reilly, M.},
\bibinfo{author}{Pinto, J. D.~A.}, \bibinfo{author}{Vispute, K.} \&
\bibinfo{author}{Lowdell, M.}
\newblock \bibinfo{title}{Manufacturing chimeric antigen receptor t cells:
issues and challenges}.
\newblock \emph{\bibinfo{journal}{Cytotherapy}} (\bibinfo{year}{2019}).
\bibitem{Dwarshuis2017}
\bibinfo{author}{Dwarshuis, N.~J.}, \bibinfo{author}{Parratt, K.},
\bibinfo{author}{Santiago-Miranda, A.} \& \bibinfo{author}{Roy, K.}
\newblock \bibinfo{title}{Cells as advanced therapeutics: State-of-the-art,
challenges, and opportunities in large scale biomanufacturing of high-quality
cells for adoptive immunotherapies}.
\newblock \emph{\bibinfo{journal}{Advanced Drug Delivery Reviews}}
\textbf{\bibinfo{volume}{114}}, \bibinfo{pages}{222--239}
(\bibinfo{year}{2017}).
\bibitem{Wang2016}
\bibinfo{author}{Wang, X.} \& \bibinfo{author}{Rivi{\`{e}}re, I.}
\newblock \bibinfo{title}{Clinical manufacturing of {CAR} t cells: foundation
of a promising therapy}.
\newblock \emph{\bibinfo{journal}{Molecular Therapy - Oncolytics}}
\textbf{\bibinfo{volume}{3}}, \bibinfo{pages}{16015} (\bibinfo{year}{2016}).
\bibitem{Piscopo2017}
\bibinfo{author}{Piscopo, N.~J.} \emph{et~al.}
\newblock \bibinfo{title}{Bioengineering solutions for manufacturing challenges
in {CAR} t cells}.
\newblock \emph{\bibinfo{journal}{Biotechnology Journal}}
\textbf{\bibinfo{volume}{13}}, \bibinfo{pages}{1700095}
(\bibinfo{year}{2017}).
\bibitem{Bashour2015}
\bibinfo{author}{Bashour, K.~T.} \emph{et~al.}
\newblock \bibinfo{title}{Functional characterization of a t cell stimulation
reagent for the production of therapeutic chimeric antigen receptor t cells}.
\newblock \emph{\bibinfo{journal}{Blood}} \textbf{\bibinfo{volume}{126}},
\bibinfo{pages}{1901--1901} (\bibinfo{year}{2015}).
\bibitem{Gendron2003}
\bibinfo{author}{Gendron, S.}, \bibinfo{author}{Couture, J.} \&
\bibinfo{author}{Aoudjit, F.}
\newblock \bibinfo{title}{Integrin $\alpha$2$\beta$1inhibits fas-mediated
apoptosis in t lymphocytes by protein phosphatase 2a-dependent activation of
the {MAPK}/{ERK} pathway}.
\newblock \emph{\bibinfo{journal}{Journal of Biological Chemistry}}
\textbf{\bibinfo{volume}{278}}, \bibinfo{pages}{48633--48643}
(\bibinfo{year}{2003}).
\bibitem{Ohtani2008}
\bibinfo{author}{Ohtani, O.} \& \bibinfo{author}{Ohtani, Y.}
\newblock \bibinfo{title}{Structure and function of rat lymph nodes}.
\newblock \emph{\bibinfo{journal}{Archives of Histology and Cytology}}
\textbf{\bibinfo{volume}{71}}, \bibinfo{pages}{69--76}
(\bibinfo{year}{2008}).
\bibitem{Boisvert2007}
\bibinfo{author}{Boisvert, M.}, \bibinfo{author}{Gendron, S.},
\bibinfo{author}{Chetoui, N.} \& \bibinfo{author}{Aoudjit, F.}
\newblock \bibinfo{title}{Alpha2beta1 integrin signaling augments t cell
receptor-dependent production of interferon-gamma in human t cells}.
\newblock \emph{\bibinfo{journal}{Molecular Immunology}}
\textbf{\bibinfo{volume}{44}}, \bibinfo{pages}{3732--3740}
(\bibinfo{year}{2007}).
\bibitem{Ben-Horin2004}
\bibinfo{author}{Ben-Horin, S.} \& \bibinfo{author}{Bank, I.}
\newblock \bibinfo{title}{The role of very late antigen-1 in immune-mediated
inflammation}.
\newblock \emph{\bibinfo{journal}{Clinical Immunology}}
\textbf{\bibinfo{volume}{113}}, \bibinfo{pages}{119--129}
(\bibinfo{year}{2004}).
\bibitem{Forget2014}
\bibinfo{author}{Forget, M.-A.} \emph{et~al.}
\newblock \bibinfo{title}{Activation and propagation of tumor-infiltrating
lymphocytes on clinical-grade designer artificial antigen-presenting cells
for adoptive immunotherapy of melanoma}.
\newblock \emph{\bibinfo{journal}{Journal of Immunotherapy}}
\textbf{\bibinfo{volume}{37}}, \bibinfo{pages}{448--460}
(\bibinfo{year}{2014}).
\bibitem{Cheung2018}
\bibinfo{author}{Cheung, A.~S.}, \bibinfo{author}{Zhang, D. K.~Y.},
\bibinfo{author}{Koshy, S.~T.} \& \bibinfo{author}{Mooney, D.~J.}
\newblock \bibinfo{title}{Scaffolds that mimic antigen-presenting cells enable
ex vivo expansion of primary {T} cells}.
\newblock \emph{\bibinfo{journal}{Nature Biotechnology}}
\textbf{\bibinfo{volume}{36}}, \bibinfo{pages}{160--169}
(\bibinfo{year}{2018}).
\bibitem{Rio2018}
\bibinfo{author}{del R{\'{\i}}o, E.~P.}, \bibinfo{author}{Miguel, M.~M.},
\bibinfo{author}{Veciana, J.}, \bibinfo{author}{Ratera, I.} \&
\bibinfo{author}{Guasch, J.}
\newblock \bibinfo{title}{Artificial 3d culture systems for t cell expansion}.
\newblock \emph{\bibinfo{journal}{{ACS} Omega}} \textbf{\bibinfo{volume}{3}},
\bibinfo{pages}{5273--5280} (\bibinfo{year}{2018}).
\bibitem{Delalat2017}
\bibinfo{author}{Delalat, B.} \emph{et~al.}
\newblock \bibinfo{title}{{3D printed lattices as an activation and expansion
platform for T cell therapy}}.
\newblock \emph{\bibinfo{journal}{Biomaterials}}
\textbf{\bibinfo{volume}{140}}, \bibinfo{pages}{58--68}
(\bibinfo{year}{2017}).
\bibitem{meyer15_immun}
\bibinfo{author}{Meyer, R.~A.} \emph{et~al.}
\newblock \bibinfo{title}{Immunoengineering: Biodegradable nanoellipsoidal
artificial antigen presenting cells for antigen specific t-cell activation
(small 13/2015)}.
\newblock \emph{\bibinfo{journal}{Small}} \textbf{\bibinfo{volume}{11}},
\bibinfo{pages}{1612--1612} (\bibinfo{year}{2015}).
\bibitem{Lambert2017}
\bibinfo{author}{Lambert, L.~H.} \emph{et~al.}
\newblock \bibinfo{title}{{Improving T Cell Expansion with a Soft Touch.}}
\newblock \emph{\bibinfo{journal}{Nano letters}} \textbf{\bibinfo{volume}{17}},
\bibinfo{pages}{821--826} (\bibinfo{year}{2017}).
\bibitem{Xu2014}
\bibinfo{author}{Xu, Y.} \emph{et~al.}
\newblock \bibinfo{title}{Closely related t-memory stem cells correlate with in
vivo expansion of car.cd19-t cells and are preserved by il-7 and il-15.}
\newblock \emph{\bibinfo{journal}{Blood}} \textbf{\bibinfo{volume}{123}},
\bibinfo{pages}{3750--3759} (\bibinfo{year}{2014}).
\bibitem{Fraietta2018}
\bibinfo{author}{Fraietta, J.~A.} \emph{et~al.}
\newblock \bibinfo{title}{Determinants of response and resistance to {CD}19
chimeric antigen receptor ({CAR}) t cell therapy of chronic lymphocytic
leukemia}.
\newblock \emph{\bibinfo{journal}{Nature Medicine}}
\textbf{\bibinfo{volume}{24}}, \bibinfo{pages}{563--571}
(\bibinfo{year}{2018}).
\bibitem{Gattinoni2011}
\bibinfo{author}{Gattinoni, L.} \emph{et~al.}
\newblock \bibinfo{title}{A human memory t cell subset with stem
cell{\textendash}like properties}.
\newblock \emph{\bibinfo{journal}{Nature Medicine}}
\textbf{\bibinfo{volume}{17}}, \bibinfo{pages}{1290--1297}
(\bibinfo{year}{2011}).
\bibitem{Gattinoni2012}
\bibinfo{author}{Gattinoni, L.}, \bibinfo{author}{Klebanoff, C.~A.} \&
\bibinfo{author}{Restifo, N.~P.}
\newblock \bibinfo{title}{{Paths to stemness: building the ultimate antitumour
T cell.}}
\newblock \emph{\bibinfo{journal}{Nature reviews. Cancer}}
\textbf{\bibinfo{volume}{12}}, \bibinfo{pages}{671--84}
(\bibinfo{year}{2012}).
\bibitem{Wang2018}
\bibinfo{author}{Wang, D.} \emph{et~al.}
\newblock \bibinfo{title}{Glioblastoma-targeted {CD}4+ {CAR} t cells mediate
superior antitumor activity}.
\newblock \emph{\bibinfo{journal}{{JCI} Insight}} \textbf{\bibinfo{volume}{3}}
(\bibinfo{year}{2018}).
\bibitem{Yang2017}
\bibinfo{author}{Yang, Y.} \emph{et~al.}
\newblock \bibinfo{title}{{TCR} engagement negatively affects {CD}8 but not
{CD}4 {CAR} t cell expansion and leukemic clearance}.
\newblock \emph{\bibinfo{journal}{Science Translational Medicine}}
\textbf{\bibinfo{volume}{9}}, \bibinfo{pages}{eaag1209}
(\bibinfo{year}{2017}).
\bibitem{Heathman2015}
\bibinfo{author}{Heathman, T. R.~J.} \emph{et~al.}
\newblock \bibinfo{title}{Expansion, harvest and cryopreservation of human
mesenchymal stem cells in a serum-free microcarrier process}.
\newblock \emph{\bibinfo{journal}{Biotechnology and Bioengineering}}
\textbf{\bibinfo{volume}{112}}, \bibinfo{pages}{1696--1707}
(\bibinfo{year}{2015}).
\bibitem{Sart2011}
\bibinfo{author}{Sart, S.}, \bibinfo{author}{Errachid, A.},
\bibinfo{author}{Schneider, Y.-J.} \& \bibinfo{author}{Agathos, S.~N.}
\newblock \bibinfo{title}{Controlled expansion and differentiation of
mesenchymal stem cells in a microcarrier based stirred bioreactor}.
\newblock \emph{\bibinfo{journal}{{BMC} Proceedings}}
\textbf{\bibinfo{volume}{5}} (\bibinfo{year}{2011}).
\end{thebibliography}

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tex/thesis.tex
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@ -48,6 +48,12 @@
\newacronym{cpp}{CPP}{critical process parameter}
\newacronym{dms}{DMS}{degradable microscaffold}
\newacronym{doe}{DOE}{design of experiments}
\newacronym{gmp}{GMP}{Good Manufacturing Practices}
\newacronym{cho}{CHO}{Chinese hamster ovary}
\newacronym{all}{ALL}{acute lymphoblastic leukemia}
\newacronym{pdms}{PDMS}{polydimethylsiloxane}
\newacronym{dc}{DC}{dendritic cell}
\newacronym{il2}{IL2}{interleukin 2}
\newcommand{\mytitle}{
\Large{
@ -234,6 +240,87 @@ quality in an industrial setting.
\chapter{introduction}
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
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 anti-CD3 and anti-CD28 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 in vivo. 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 in vivo 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.
% TODO mention the Cloudz stuff that's in my presentation
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 in vivo. 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.
% TODO probably need to address some of the modeling stuff here as well
This thesis describes a novel degradable microscaffold-based method derived from
porous microcarriers functionalized with anti-CD3 and anti-CD28 \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) {\#}[Purcell
documentation]. 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 in vivo 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.
\section*{overview}
Insert overview here
@ -310,7 +397,7 @@ bla bla
\chapter{References}
\renewcommand{\section}[2]{} % noop the original bib section header
\bibliography{../proposal}
\bibliography{references}
\bibliographystyle{naturemag}