diff --git a/tex/thesis.tex b/tex/thesis.tex index 412216a..80dca44 100644 --- a/tex/thesis.tex +++ b/tex/thesis.tex @@ -4522,7 +4522,7 @@ porcine-derived collagen, which itself is not \gls{gmp}-compliant due to its non-human animal origins. However, using any other source of collagen should work so long as the structure of the microcarriers remains relatively similar and it has lysine groups that can react with the \gls{snb} to attach \gls{stp} -and \glspl{mab}. Obviously these would need to be tested and verified, but they +and \glspl{mab}. Obviously these would need to be tested and verified, but these should not be insurmountable. Furthermore, the \gls{mab} binding step requires \gls{bsa} to prevent adsorption to the non-polar polymer walls of the reaction tubes. A human carrier protein such as \gls{hsa} could be used in its place to @@ -4531,7 +4531,7 @@ 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\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 +\gls{bsa} from the process. The \gls{stp} and \glspl{mab} in this work 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 @@ -4545,7 +4545,12 @@ as well as it does. Several broad areas remain to be investigated, including the role of the increased cytokine output (including \il{15} which was explored to some extent in this work), the role of cells on the interior of the \gls{dms} relative to those outside the \gls{dms}, and the role of the physical surface -properties of the \gls{dms} (including the morphology and the stiffness). +properties of the \gls{dms} (including the morphology and the stiffness). One +plausible hypothesis to be tested is that the bumpy microcarrier surface is more +like that of an \gls{apc}, which enhances immunological synapse formation and +thus activation. Another related hypothesis is that the signal strength is +lower than the beads, which leads to increased proliferation, less exhaustion, +and by extension more memory. \subsection{additional ligands and signals on the DMSs} @@ -4553,22 +4558,23 @@ In this work we only explored the use of \acd{3} and \acd{28} \glspl{mab} coated on the surface of the \gls{dms}. The chemistry used for the \gls{dms} is very general, and any molecule or protein that could be engineered with a biotin ligand could be attached without any further modification. There are many other -ligands that could have profound effects on the expansion and quality of T cells -which may be utilized. The simplest next step is to simply vary the ratio of -\acd{3} and \acd{28} signal. Another obvious example is to attach -\il{15}/\il{15R$\upalpha$} complexes to the surface to mimic \textit{trans} -presentation from other cell types\cite{Stonier2010}. Other adhesion ligands or -peptides such as GFOGER could be used to stimulate T cells and provide more -motility on the \glspl{dms}\cite{Stephan2014}. Finally, viral delivery systems -could theoretically be attached to the \gls{dms}, greatly simplifying the -transduction step. +ligands (in addition to integrin-binding domains and \il{15} complexes as +described at the end of \cref{aim2b}) that could have profound effects on the +expansion and quality of T cells which may be utilized. The simplest next step +is to simply vary the ratio of \acd{3} and \acd{28} signal. Another obvious +example is to attach \il{15}/\il{15R$\upalpha$} complexes to the surface to +mimic \textit{trans} presentation from other cell types\cite{Stonier2010}. Other +adhesion ligands or peptides such as GFOGER could be used to stimulate T cells +and provide more motility on the \glspl{dms}\cite{Stephan2014}. Finally, viral +delivery systems could theoretically be attached to the \gls{dms}, greatly +simplifying the transduction step. \subsection{assessing performance using unhealthy donors} All the work presented in this dissertation was performed using healthy donors. This was mostly due to the fact that it was much easier to obtain healthy donor cells and was much easier to control. However, it is indisputable that the most -relevant test cases of the \gls{dms} will be for unhealthy patient T cells, at +relevant test cases of the \glspl{dms} will be for unhealthy patient T cells, at least in the case of autologous therapies. In particular, it will be interesting to see how the \gls{dms} performs when assessed head-to-head with bead-based expansion technology given that even in healthy donors, we observed the