diff --git a/tex/thesis.tex b/tex/thesis.tex index 7330736..11a0a76 100644 --- a/tex/thesis.tex +++ b/tex/thesis.tex @@ -68,6 +68,7 @@ \newacronym{pbs}{PBS}{phosphate buffered saline} \newacronym{bca}{BCA}{bicinchoninic acid assay} \newacronym{bsa}{BSA}{bovine serum albumin} +\newacronym{hsa}{HSA}{human serum albumin} \newacronym{stp}{STP}{streptavidin} \newacronym{stppe}{STP-PE}{streptavidin-phycoerythrin} \newacronym{snb}{SNB}{sulfo-nhs-biotin} @@ -3476,15 +3477,64 @@ In \cref{aim3} we determined that the \glspl{dms} expand T cells that also performed better than beads \invivo{}. In the first experiment we performed, the results were very clearly in favor of the \glspl{dms}. In the second experiment, even the \gls{dms} group failed to fully control the tumor burden, but this is -not surprising given the low \ptcarp{} across all groups. Also, despite this, the -\gls{dms} group appeared to control the tumor better on average for early, mid, -and late T cell harvesting timepoints. It was not clear if this effect was due -to increased \cdp{} or overall increased fitness of the \gls{dms}-expanded T +not surprising given the low \ptcarp{} across all groups. Also, despite this, +the \gls{dms} group appeared to control the tumor better on average for early, +mid, and late T cell harvesting timepoints. It was not clear if this effect was +due to increased \cdp{} or overall increased fitness of the \gls{dms}-expanded T cells given their higher expansion rate. The \ptmemp{} did not seem to be a factor given that it was nearly the same in the first experiment between -\gls{dms} and bead groups despite the clear advantage seen in the \gls{dms} group. +\gls{dms} and bead groups despite the clear advantage seen in the \gls{dms} +group. -\section{future work} +\section{future directions} + +There are several important next steps to perform with this work: + +\subsection{Translation to GMP process} + +While this work was done with translatability and \gls{qc} in mind, an important +feature that is missing from the process currently is the use of \gls{gmp} +methods and materials. The microcarriers themselves are made from +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 +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 +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 +replaced. The \gls{snb} is a synthetic small molecule and thus does not have any +animal-origin concerns. + +\subsection{Mechanistic investigation} + +% why do the dms work? +% can we put anything on the dms to enhance their potency? + +\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 +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 +\gls{dms} platform to work where the beads failed +(\cref{fig:dms_exp_fold_change}). + +\subsection{translation to bioreactors} + +% use dms in non-static bioreactors such as wave by first activating in a static +% environment \onecolumn \clearpage