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@ -2475,6 +2475,75 @@ CONCLUSIONS: We developed a simplified, semi-closed system for the initial selec
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publisher = {Springer Science and Business Media {LLC}},
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}
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@Article{Kamholz2001,
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author = {Kamholz, A. E. and Schilling, E. A. and Yager, P.},
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journal = {Biophysical journal},
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title = {Optical measurement of transverse molecular diffusion in a microchannel.},
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year = {2001},
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issn = {0006-3495},
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month = apr,
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pages = {1967--1972},
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volume = {80},
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abstract = {Quantitative analysis of molecular diffusion is a necessity for the efficient design of most microfluidic devices as well as an important biophysical method in its own right. This study demonstrates the rapid measurement of diffusion coefficients of large and small molecules in a microfluidic device, the T-sensor, by means of conventional epifluorescence microscopy. Data were collected by monitoring the transverse flux of analyte from a sample stream into a second stream flowing alongside it. As indicated by the low Reynolds numbers of the system (< 1), flow is laminar, and molecular transport between streams occurs only by diffusion. Quantitative determinations were made by fitting data with predictions of a one-dimensional model. Analysis was made of the flow development and its effect on the distribution of diffusing analyte using a three-dimensional modeling software package. Diffusion coefficients were measured for four fluorescently labeled molecules: fluorescein-biotin, insulin, ovalbumin, and streptavidin. The resulting values differed from accepted results by an average of 2.4%. Microfluidic system parameters can be selected to achieve accurate diffusion coefficient measurements and to optimize other microfluidic devices that rely on precise transverse transport of molecules.},
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chemicals = {Biotin},
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citation-subset = {IM},
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completed = {2001-06-21},
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country = {United States},
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doi = {10.1016/S0006-3495(01)76166-8},
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issn-linking = {0006-3495},
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issue = {4},
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keywords = {Biochemistry, instrumentation; Biotin, pharmacology; Computer Simulation; Diffusion; Microscopy, Fluorescence, instrumentation, methods; Models, Theoretical},
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nlm-id = {0370626},
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owner = {NLM},
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pii = {S0006-3495(01)76166-8},
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pmc = {PMC1301385},
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pmid = {11259309},
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pubmodel = {Print},
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pubstate = {ppublish},
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revised = {2018-11-13},
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}
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@Article{Niether2020,
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author = {Doreen Niether and Mona Sarter and Bernd W. Koenig and Jörg Fitter and Andreas M. Stadler and Simone Wiegand},
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journal = {Polymers},
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title = {Thermophoresis: The Case of Streptavidin and Biotin},
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year = {2020},
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month = {feb},
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number = {2},
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pages = {376},
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volume = {12},
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doi = {10.3390/polym12020376},
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publisher = {{MDPI} {AG}},
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}
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@Article{Kerwin2008,
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author = {Kerwin, Bruce A.},
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journal = {Journal of pharmaceutical sciences},
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title = {Polysorbates 20 and 80 used in the formulation of protein biotherapeutics: structure and degradation pathways.},
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year = {2008},
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issn = {1520-6017},
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month = aug,
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pages = {2924--2935},
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volume = {97},
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abstract = {Polysorbates 20 and 80 (Tween 20 and Tween 80) are used in the formulation of biotherapeutic products for both preventing surface adsorption and as stabilizers against protein aggregation. The polysorbates are amphipathic, nonionic surfactants composed of fatty acid esters of polyoxyethylene sorbitan being polyoxyethylene sorbitan monolaurate for polysorbate 20 and polyoxyethylene sorbitan monooleate for polysorbate 80. The polysorbates used in the formulation of biopharmaceuticals are mixtures of different fatty acid esters with the monolaurate fraction of polysorbate 20 making up only 40-60% of the mixture and the monooleate fraction of polysorbate 80 making up >58% of the mixture. The polysorbates undergo autooxidation, cleavage at the ethylene oxide subunits and hydrolysis of the fatty acid ester bond. Autooxidation results in hydroperoxide formation, side-chain cleavage and eventually formation of short chain acids such as formic acid all of which could influence the stability of a biopharmaceutical product. Oxidation of the fatty acid moiety while well described in the literature has not been specifically investigated for polysorbate. This review focuses on the chemical structure of the polysorbates, factors influencing micelle formation and factors and excipients influencing stability and degradation of the polyoxyethylene and fatty acid ester linkages.},
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chemicals = {Polysorbates, Proteins, Surface-Active Agents},
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citation-subset = {IM},
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completed = {2008-11-04},
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country = {United States},
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doi = {10.1002/jps.21190},
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issn-linking = {0022-3549},
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issue = {8},
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keywords = {Chemistry, Pharmaceutical; Molecular Structure; Oxidation-Reduction; Polysorbates, chemistry; Proteins, administration & dosage, chemical synthesis, therapeutic use; Surface Tension; Surface-Active Agents, chemistry},
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nlm-id = {2985195R},
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owner = {NLM},
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pii = {S0022-3549(16)32657-0},
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pmid = {17973307},
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pubmodel = {Print},
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pubstate = {ppublish},
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references = {91},
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revised = {2008-07-28},
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}
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@Comment{jabref-meta: databaseType:bibtex;}
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@Comment{jabref-meta: grouping:
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155
tex/thesis.tex
155
tex/thesis.tex
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@ -485,8 +485,8 @@ such as bioreactors.
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% TODO probably need to address some of the modeling stuff here as well
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This thesis describes a novel degradable microscaffold-based method derived from
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porous microcarriers functionalized with \acd{3} and \acd{28} \glspl{mab}
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for use in T cell expansion cultures. Microcarriers have historically been used
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porous microcarriers functionalized with \acd{3} and \acd{28} \glspl{mab} for
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use in T cell expansion cultures. Microcarriers have historically been used
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throughout the bioprocess industry for adherent cultures such as stem cells and
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\gls{cho} cells, but not with suspension cells such as T
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cells\cite{Heathman2015, Sart2011}. The microcarriers chosen to make the DMSs in
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@ -499,17 +499,17 @@ larger contact area for T cells to interact with the \glspl{mab} relative to
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beads; this may better emulate the large contact surface area that occurs
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between T cells and \glspl{dc}. These microcarriers are readily available in
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over 30 countries and are used in an FDA fast-track-approved combination retinal
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pigment epithelial cell product (Spheramine, Titan Pharmaceuticals) {\#}[Purcell
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documentation]. This regulatory history will aid in clinical translation. We
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show that compared to traditional microbeads, \gls{dms}-expanded T cells not
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only provide superior expansion, but consistently provide a higher frequency of
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naïve/memory and CD4 T cells (CCR7+CD62L+) across multiple donors. We also
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demonstrate functional cytotoxicity using a CD19 \gls{car} and a superior
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performance, even at a lower \gls{car} T cell dose, of \gls{dms}-expanded
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\gls{car}-T cells \invivo{} in a mouse xenograft model of human B cell
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\gls{all}. Our results indicate that \glspl{dms} provide a robust and scalable
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platform for manufacturing therapeutic T cells with higher naïve/memory
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phenotype and more balanced CD4+ T cell content.
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pigment epithelial cell product (Spheramine, Titan
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Pharmaceuticals)\cite{purcellmain}. This regulatory history will aid in clinical
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translation. We show that compared to traditional microbeads, \gls{dms}-expanded
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T cells not only provide superior expansion, but consistently provide a higher
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frequency of naïve/memory and CD4 T cells (CCR7+CD62L+) across multiple donors.
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We also demonstrate functional cytotoxicity using a CD19 \gls{car} and a
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superior performance, even at a lower \gls{car} T cell dose, of
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\gls{dms}-expanded \gls{car}-T cells \invivo{} in a mouse xenograft model of
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human B cell \gls{all}. Our results indicate that \glspl{dms} provide a robust
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and scalable platform for manufacturing therapeutic T cells with higher
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naïve/memory phenotype and more balanced CD4+ T cell content.
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\section*{hypothesis}
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@ -1569,10 +1569,9 @@ diffusion coefficient of \gls{stp} in water. This model was given by
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\item $n$ is the number of microcarriers in the reaction volume
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\end{itemize}
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% TODO cite the diffusion rate of stp
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The diffusion rate of \gls{stp} was assumed to be
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\SI{3.89e-7}{\cm\squared\per\second} {\#}{diffusion rate citation}. Since all
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but $\beta$ was known, the experimental data was fit using these equations using
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\SI{6.2e-7}{\cm\squared\per\second}\cite{Kamholz2001}. Since all but $\beta$ was
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known, the experimental data was fit using these equations using
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\inlinecode{ode45} in MATLAB and least squares as the fitting error.
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% TODO this diffusion rate isn't actually reflected in the code
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@ -1582,6 +1581,9 @@ These equations were then used analogously to describe the reaction profile of
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% METHOD add the equation governing the washing steps
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The diffusion coefficient used for biotin was
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\SI{5e-6}{\cm\squared\per\second}\cite{Niether2020}
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\subsection{Luminex Analysis}\label{sec:luminex_analysis}
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Luminex was performed using a \product{ProcartaPlex kit}{\thermo}{custom} for
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@ -1685,25 +1687,25 @@ context of pure error). Statistical significance was evaluated at $\upalpha$ =
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Two types of gelatin-based microcariers, \gls{cus} and \gls{cug}, were
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covalently conjugated with varying concentration of \gls{snb} and then coated
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with \gls{stp} and \glspl{mab} to make \glspl{dms}. Aside from slight
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differences in swelling ratio and crosslinking chemistry {\#}[Purcell
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documentation], the properties of \gls{cus} and \gls{cug} were the same
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(\cref{tab:carrier_props}). We chose to continue with the \gls{cus}-based
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\glspl{dms}, which showed higher overall \gls{stp} binding compared to
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\gls{cug}-based \glspl{dms} (\cref{fig:cug_vs_cus}). We showed that by varying
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the concentration of \gls{snb}, we were able to precisely control the amount of
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attached biotin (\cref{fig:biotin_coating}), mass of attached \gls{stp}
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differences in swelling ratio and crosslinking chemistry\cite{purcellmain} the
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properties of \gls{cus} and \gls{cug} were the same (\cref{tab:carrier_props}).
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We chose to continue with the \gls{cus}-based \glspl{dms}, which showed higher
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overall \gls{stp} binding compared to \gls{cug}-based \glspl{dms}
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(\cref{fig:cug_vs_cus}). We showed that by varying the concentration of
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\gls{snb}, we were able to precisely control the amount of attached biotin
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(\cref{fig:biotin_coating}), mass of attached \gls{stp}
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(\cref{fig:stp_coating}), and mass of attached \glspl{mab}
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(\cref{fig:mab_coating}). Furthermore, we showed that the microcarriers were
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evenly coated with \gls{stp} on the surface and throughout the interior as
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evidenced by the presence of biotin-binding sites occupied with \gls{stp}-\gls{fitc}
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on the microcarrier surfaces after the \gls{stp}-coating step
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(\cref{fig:stp_carrier_fitc}). Finally, we confirmed that biotinylated
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evidenced by the presence of biotin-binding sites occupied with
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\gls{stp}-\gls{fitc} on the microcarrier surfaces after the \gls{stp}-coating
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step (\cref{fig:stp_carrier_fitc}). Finally, we confirmed that biotinylated
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\glspl{mab} were bound to the \glspl{dms} by staining either \gls{stp} or
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\gls{stp} and \gls{mab}-coated carriers with \antim{\gls{igg}-\gls{fitc}} and imaging
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on a confocal microscope (\cref{fig:mab_carrier_fitc}). Taking this together, we
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noted that the maximal \gls{mab} binding capacity occurred near \SI{50}{\nmol}
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biotin input (which corresponded to \SI{2.5}{\nmol\per\mg\of{\dms}}) thus we
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used this in subsequent processes.
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\gls{stp} and \gls{mab}-coated carriers with \antim{\gls{igg}-\gls{fitc}} and
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imaging on a confocal microscope (\cref{fig:mab_carrier_fitc}). Taking this
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together, we noted that the maximal \gls{mab} binding capacity occurred near
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\SI{50}{\nmol} biotin input (which corresponded to
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\SI{2.5}{\nmol\per\mg\of{\dms}}) thus we used this in subsequent processes.
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% TODO flip the rows of this figure (right now the text is backward)
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\begin{figure*}[ht!]
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@ -1872,7 +1874,7 @@ using the \gls{bca} assay to indirectly quantify the amount of attached
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(\cref{fig:dms_stp_per_time}). Assuming a quasi-steady-state paradigm, we used
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this experimental binding data to fit a continuous model for the \gls{stp}
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binding reaction. Using the diffusion rate of the \gls{stp}, we then calculated
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the effective diffusivity of the microcarriers to be {\#}.
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the effective diffusivity of the microcarriers to be 0.2.
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Using this effective diffusivity and the known diffusion coefficient of a
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\gls{mab} protein in water, we calculated predict the binding of \glspl{mab} per
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@ -2002,9 +2004,9 @@ membrane. When staining for these two markers and assessing via flow cytometry,
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we observe that the \gls{dms}-expanded T cells have lower frequencies of
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apoptotic and necrotic cells (\cref{fig:apoptosis_annV}). Furthermore, we
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stained our cultures with CellEvent dye, which is an indicator of \gls{cas37},
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which is activated in apoptotic cells {\#}{cas37 activation}. In line with the
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\gls{pi}/\gls{anv} results, we observed that the \gls{dms} T cells had lower
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frequency of \gls{cas37} expression, indicating less apoptosis for our method
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which is activated in apoptotic cells. In line with the \gls{pi}/\gls{anv}
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results, we observed that the \gls{dms} T cells had lower frequency of
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\gls{cas37} expression, indicating less apoptosis for our method
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(\cref{fig:apoptosis_cas}). Finally, we lysed our cells and stained for
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\gls{bcl2}, which is also upregulated in apoptosis. In this case, some (but not
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all) of the bead-expanded cultures showed higher \gls{bcl2} expression, which
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@ -3642,8 +3644,8 @@ them to grow better in the \gls{dms} system.
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We tested this hypothesis by adding blocking \glspl{mab} against \gls{a2b1}
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and/or \gls{a2b2} to running T cell cultures activated using the \glspl{dms}.
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These block \glspl{mab} were added at day 6 of culture when \gls{a2b1} and
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\gls{a2b2} were known to be expressed {\#}. We found that the fold expansion was
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identical in all the blocked groups vs the unblocked control group
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\gls{a2b2} were known to be expressed\cite{Hemler1990}. We found that the fold
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expansion was identical in all the blocked groups vs the unblocked control group
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(\cref{fig:inegrin_1_fc}). Furthermore, we observed that the \ptmemp{} (total
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and across the CD4/CD8 compartments) was not significantly different between any
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of the groups (\cref{fig:inegrin_1_mem,tab:integrin_1_reg}). We also noted that
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@ -3808,13 +3810,13 @@ receptors may simply be irrelevant for our system.
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On the first point, we did not verify that these \glspl{mab} indeed blocked the
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receptor we were targeting. There has been evidence from other groups that these
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particular clones work at the concentrations we used {\#}. This does not
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necessarily mean that the \glspl{mab} we obtained were functional in blocking
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their intended targets (although they were from a reputable manufacturer, \bl).
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Furthermore, we can safely rule out the possibility that the \glspl{mab} never
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reached their targets, as they were added immediately after the T cells were
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resuspended as required for cell counting, hence their resting clustered state
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was disrupted.
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particular clones work at the concentrations we used\cite{MirandaCarus2005}.
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This does not necessarily mean that the \glspl{mab} we obtained were functional
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in blocking their intended targets (although they were from a reputable
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manufacturer, \bl). Furthermore, we can safely rule out the possibility that the
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\glspl{mab} never reached their targets, as they were added immediately after
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the T cells were resuspended as required for cell counting, hence their resting
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clustered state was disrupted.
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% TODO define Bite
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On the second point, the collagen domains may not even be relevant to our system
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@ -4046,15 +4048,15 @@ case of beads (\cref{fig:mouse_dosing_qc_mem}).
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\subsection{Beads and DMSs perform similarly at earlier timepoints}
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We then asked how T cells harvested using either beads or \gls{dms} performed
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when harvested at earlier timepoints {\#}{levine paper with early timepoints}.
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We performed the same experiments as described in
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\cref{fig:mouse_dosing_overview} with the modification that T cells were only
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grown and harvested after \SI{6}{\day}, \SI{10}{\day}, or \SI{14}{\day} of
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expansion (\cref{fig:mouse_timecourse_overview}). T cells were frozen after
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harvest, and all timepoints were thawed at the same time prior to injection. The
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dose of T cells injected was \num{1.25e6} cells per mouse (the same as the high
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dose in the first experiment). All other characteristics of the experiment were
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the same.
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when harvested at earlier timepoints\cite{Ghassemi2018}. We performed the same
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experiments as described in \cref{fig:mouse_dosing_overview} with the
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modification that T cells were only grown and harvested after \SI{6}{\day},
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\SI{10}{\day}, or \SI{14}{\day} of expansion
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(\cref{fig:mouse_timecourse_overview}). T cells were frozen after harvest, and
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all timepoints were thawed at the same time prior to injection. The dose of T
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cells injected was \num{1.25e6} cells per mouse (the same as the high dose in
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the first experiment). All other characteristics of the experiment were the
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same.
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\begin{figure*}[ht!]
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\begingroup
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@ -4083,7 +4085,7 @@ untransduced cells. The \pthp{} of the harvested T cells was higher overall in
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(\cref{fig:mouse_timecourse_qc_cd4}). The \ptmemp{} was similar at day 6
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between bead and \gls{dms} groups but the \gls{dms} group had higher \ptmemp{}
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at day 14 despite the overall \ptmemp{} decreasing with time as shown elsewhere
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(\cref{fig:mouse_timecourse_qc_mem}) {\#}{levine paper mem over time}.
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(\cref{fig:mouse_timecourse_qc_mem})\cite{Ghassemi2018}.
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\begin{figure*}[ht!]
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\begingroup
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@ -4265,17 +4267,17 @@ for the patient, while also minimizing cost for the manufacturer. Second,
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\glspl{cqa} can be used to define process control schemes as well as release
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criteria. Process control, and with it the ability to predict future outcomes
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based on data obtained at the present, is highly important for cell therapies
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given that batch failures are extremely expensive {\#}, and predicting a batch
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failure would allow manufacturers to restart the batch in a timely manner
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without wasting resources. Furthermore, \glspl{cqa} can be used to define what a
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`good' vs `bad' product is, which will important help anticipate dosing and
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followup procedures in the clinic if the T cells are administered. In the aim,
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we cannot claim to have found the ultimate set of \glspl{cqa} and \glspl{cpp},
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as we used tissue culture plates instead of a bioreactor and we only used one
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donor. However, we have indeed outlined a process that others may use to find
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these for their process. In particular, the 2-phase modeling process we used
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(starting with a \gls{doe} and collecting data longitudinally) is a strategy
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that manufacturers can easily implement. Also, collecting secretome and
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given that batch failures are extremely expensive\cite{Harrison2019}, and
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predicting a batch failure would allow manufacturers to restart the batch in a
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timely manner without wasting resources. Furthermore, \glspl{cqa} can be used to
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define what a `good' vs `bad' product is, which will important help anticipate
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dosing and followup procedures in the clinic if the T cells are administered. In
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the aim, we cannot claim to have found the ultimate set of \glspl{cqa} and
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\glspl{cpp}, as we used tissue culture plates instead of a bioreactor and we
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only used one donor. However, we have indeed outlined a process that others may
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use to find these for their process. In particular, the 2-phase modeling process
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we used (starting with a \gls{doe} and collecting data longitudinally) is a
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strategy that manufacturers can easily implement. Also, collecting secretome and
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metabolome is easily generalized to any setting and to most bioreactors and
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expansion systems, as they can be obtained with relatively inexpensive equipment
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(Luminex assay, benchtop \gls{nmr}, etc) without disturbing the cell culture.
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@ -4285,12 +4287,13 @@ to control and optimize the \gls{dms} system. We determined that altering the
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\gls{dms} concentration temporally has profound effects on the phenotype and
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expansion rate. This agrees with other data we obtained in \cref{aim2a} and with
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what others have generally reported about signal strength and T cell
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differentiation {\#}. We did not find any mechanistic relationship between
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either integrin signaling or \gls{il15} signaling. In the case of the former, it
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may be more likely that the \glspl{dms} surfaces are saturated to the point of
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sterically hindering any integrin interactions with the collagen surface. In the
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case of \gls{il15} more experiments likely need to be done in order to plausibly
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rule out this mechanism and/or determine if it is involved at all.
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differentiation\cite{Gattinoni2012}. We did not find any mechanistic
|
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relationship between either integrin signaling or \gls{il15} signaling. In the
|
||||
case of the former, it may be more likely that the \glspl{dms} surfaces are
|
||||
saturated to the point of sterically hindering any integrin interactions with
|
||||
the collagen surface. In the case of \gls{il15} more experiments likely need to
|
||||
be done in order to plausibly rule out this mechanism and/or determine if it is
|
||||
involved at all.
|
||||
|
||||
% TODO make this tighter and cite paper showing that this makes at least some
|
||||
% sense
|
||||
|
@ -4327,11 +4330,11 @@ tubes. A human carrier protein such as \gls{hsa} could be used in its place to
|
|||
eliminate the non-human animal origin material, but this could be much more
|
||||
expensive. Alternatively, the use of protein could be replaced altogether by a
|
||||
non-ionic detergent such as Tween-20 or Tween-80, which are already used for
|
||||
commercial \gls{mab} formulations for precisely this purpose {\#}. Validating
|
||||
the process with Tween would be the best next step to eliminate \gls{bsa} from
|
||||
the process. The \gls{stp} and \glspl{mab} in this process were not
|
||||
\gls{gmp}-grade; however, they are commonly used in clinical technology such as
|
||||
dynabeads and thus the research-grade proteins used here could be easily
|
||||
commercial \gls{mab} formulations for precisely this purpose\cite{Kerwin2008}.
|
||||
Validating the process with Tween would be the best next step to eliminate
|
||||
\gls{bsa} from the process. The \gls{stp} and \glspl{mab} in this process were
|
||||
not \gls{gmp}-grade; however, they are commonly used in clinical technology such
|
||||
as dynabeads and thus the research-grade proteins used here could be easily
|
||||
replaced. The \gls{snb} is a synthetic small molecule and thus does not have any
|
||||
animal-origin concerns.
|
||||
|
||||
|
|
Loading…
Reference in New Issue