ndwarshuis
/
phd_thesis
1
0
Fork 0
Code Issues Pull Requests Packages Projects Releases Wiki Activity

ADD bcma car section

This commit is contained in:
Nathan Dwarshuis 2021-08-03 11:43:43 -04:00
parent 530339f971
commit 8415b9519d
2 changed files with 68 additions and 27 deletions
Show Stats Download Patch File Download Diff File Expand all files Collapse all files

14
tex/references.bib
View File

@ -2200,7 +2200,7 @@ CONCLUSIONS: We developed a simplified, semi-closed system for the initial selec
country = {United States},
issn-linking = {1538-4047},
issue = {6},
keywords = {Acute Disease; Animals; Cell Line, Tumor; Cell Transplantation; Clone Cells; Collagen, metabolism; Culture Media, Conditioned, analysis; Cytokines, immunology; Drug Combinations; Infections, blood, immunology; Interferon-gamma, analysis, immunology; Interleukin-10, deficiency; Laminin, metabolism; Listeria monocytogenes, pathogenicity; Lymphocytic choriomeningitis virus, pathogenicity; Melanoma, Experimental, blood, immunology, pathology; Mice; Mice, Inbred C57BL; Mice, Knockout; Models, Biological; Neovascularization, Pathologic; Proteoglycans, metabolism; Th1 Cells, immunology; Time Factors; Toxoplasma, pathogenicity; Toxoplasmosis; Vascular Endothelial Growth Factor A, analysis, genetics, metabolism},
keywords = {Acute Disease; Animals; Cell Line, Tumor; Cell Transplantation; Clone Cells; Collagen, metabolism; Culture Media, Conditioned, analysis; Cytokines, immunology; Drug Combinations; Infections, blood, immunology; Interferon-gamma, analysis, immunology; Interleukin-10, deficiency; Laminin, metabolism; Listeria monocytogenes, pathogenicity; Lymphocytic choriomeningitis virus, pathogenicity; Melanoma, Experimental, immunology, pathology; Mice; Mice, Inbred C57BL; Mice, Knockout; Models, Biological; Neovascularization, Pathologic; Proteoglycans, metabolism; Th1 Cells, immunology; Time Factors; Toxoplasma, pathogenicity; Toxoplasmosis; Vascular Endothelial Growth Factor A, genetics, metabolism},
nlm-id = {101137842},
owner = {NLM},
pii = {557},
@ -2463,6 +2463,18 @@ CONCLUSIONS: We developed a simplified, semi-closed system for the initial selec
url = {https://www.ebook.de/de/product/8324352/wu\_hamada\_experiments\_2e.html},
}
@Article{Lam2020,
author = {Norris Lam and Nathan D. Trinklein and Benjamin Buelow and George H. Patterson and Namrata Ojha and James N. Kochenderfer},
journal = {Nature Communications},
title = {Anti-{BCMA} chimeric antigen receptors with fully human heavy-chain-only antigen recognition domains},
year = {2020},
month = {jan},
number = {1},
volume = {11},
doi = {10.1038/s41467-019-14119-9},
publisher = {Springer Science and Business Media {LLC}},
}
@Comment{jabref-meta: databaseType:bibtex;}
@Comment{jabref-meta: grouping:

81
tex/thesis.tex
View File

@ -162,6 +162,7 @@
\newacronym{talen}{TALEN}{transcription activator-like effector nuclease}
\newacronym{qbd}{QbD}{quality-by-design}
\newacronym{aws}{AWS}{amazon web services}
\newacronym{qpcr}{qPCR}{quantitative polymerase chain reaction}
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%
% SI units for uber nerds
@ -243,6 +244,7 @@
\newcommand{\catnum}[2]{(#1, #2)}
\newcommand{\product}[3]{#1 \catnum{#2}{#3}}
\newcommand{\thermo}{Thermo Fisher}
\newcommand{\sigald}{Sigma Aldrich}
\newcommand{\miltenyi}{Miltenyi Biotech}
\newcommand{\bl}{Biolegend}
\newcommand{\bd}{Becton Dickenson}
@ -743,7 +745,7 @@ material used for tissue culture flasks and dishes in general) with no
modification (SoloHill Plastic, Nunc Biosilon), with a cationic surface charge
(SoloHill Hillex) or coated with an \gls{ecm} protein such as collagen (SoloHill
Fact III). Other base materials have been used such as dextran (GE Healthcare
Cytodex), cellulose (GE Healthcare Cytopore), and glass (Sigma Aldrich G2767),
Cytodex), cellulose (GE Healthcare Cytopore), and glass (\sigald{} G2767),
all with none or similar surface modifications. Additionally, some microcarriers
such as \gls{cus} and \gls{cug} are composed entirely out of protein (in these
cases, porcine collagen) which also allows the microcarriers to be enzymatically
@ -1281,7 +1283,7 @@ two washes were done and the agitation time was \SI{30}{\minute}. Biotinylated
\glspl{mab} against human CD3 \catnum{\bl}{317320} and CD28 \catnum{\bl}{302904}
were combined in a 1:1 mass ratio and added to the carriers at
\SI{0.2}{\ug\of{\ab}\per\mg\of{\dms}}. Along with the \glspl{mab}, sterile
\product{\gls{bsa}}{Sigma}{A9576} was added to a final concentration of
\product{\gls{bsa}}{\sigald}{A9576} was added to a final concentration of
\SI{2}{\percent} in order to prevent non-specific binding of the antibodies to
the reaction tubes. \glspl{mab} were allowed to bind to the carriers for
\SI{60}{\minute} with \SI{700}{\rpm} agitation. After binding, supernatants were
@ -1299,12 +1301,12 @@ was then manually counted to obtain a concentration. Surface area for
\subsection{dms quality control assays}
Biotin was quantified using the \product{\gls{haba} assay}{Sigma}{H2153-1VL}. In
the case of quantifying \gls{snb} prior to adding it to the microcarriers, the
sample volume was quenched in a 1:1 volumetric ratio with \SI{1}{\molar} NaOH
and allowed to react for \SI{1}{\minute} in order to prevent the reactive ester
linkages from binding to the avidin proteins in the \gls{haba}/avidin premix.
All quantifications of \gls{haba} were performed on an Eppendorf D30
Biotin was quantified using the \product{\gls{haba} assay}{\sigald}{H2153-1VL}.
In the case of quantifying \gls{snb} prior to adding it to the microcarriers,
the sample volume was quenched in a 1:1 volumetric ratio with \SI{1}{\molar}
NaOH and allowed to react for \SI{1}{\minute} in order to prevent the reactive
ester linkages from binding to the avidin proteins in the \gls{haba}/avidin
premix. All quantifications of \gls{haba} were performed on an Eppendorf D30
Spectrophotometer using \product{\SI{70}{\ul} cuvettes}{BrandTech}{759200}. The
extinction coefficient at \SI{500}{\nm} for \gls{haba}/avidin was assumed to be
\SI{34000}{\per\cm\per\molar}.
@ -1457,25 +1459,52 @@ Briefly, cells were washed once and stained with \product{biotinylated
BD Accuri. Readout was percent \gls{pe}+ cells as compared to secondary controls
(\gls{pe}-\gls{stp} with no \gls{ptnl}).
% TODO add BCMA-CAR stuff
\subsection{car plasmid and lentiviral transduction}
The anti-CD19-CD8-CD137-CD3z \gls{car} with the EF1$\upalpha$
The anti-CD19-CD8-CD137-CD3$\upzeta$ \gls{car} with the EF1$\upalpha$
promotor\cite{Milone2009} was synthesized (Aldevron) and subcloned into a
\product{FUGW}{Addgene}{14883} kindly provided by the Emory Viral Vector Core.
Lentiviral vectors were synthesized by the Emory Viral Vector Core or the
Cincinnati Children's Hospital Medical Center Viral Vector Core. To transduce
primary human T cells, \product{retronectin}{Takara}{T100A} was coated onto
non-TC treated 96 well plates and used to immobilize lentiviral vector particles
according to the manufacturer's instructions. Briefly, retronectin solution was
adsorbed overnight at \SI{4}{\degreeCelsius} and blocked the next day using
\gls{bsa}. Prior to transduction, lentiviral supernatant was spinoculated at
\SI{2000}{\gforce} for \SI{2}{\hour} at \SI{4}{\degreeCelsius}. T cells were
activated in 96 well plates using beads or \glspl{dms} for \SI{24}{\hour}, and
then cells and beads/\glspl{dms} were transferred onto lentiviral vector coated
plates and incubated for another \SI{24}{\hour}. Cells and beads/\glspl{dms}
were removed from the retronectin plates using vigorous pipetting and
transferred to another 96 well plate wherein expansion continued.
Cincinnati Children's Hospital Medical Center Viral Vector Core. RNA titer was
calculated using a \product{Lenti-X \gls{qpcr} titer kit}{Takara}{631235}. To
transduce primary human T cells, \product{retronectin}{Takara}{T100A} was coated
onto non-TC treated 96 well plates and used to immobilize lentiviral vector
particles according to the manufacturer's instructions. Briefly, retronectin
solution was adsorbed overnight at \SI{4}{\degreeCelsius} and blocked the next
day using \gls{bsa}. Prior to transduction, lentiviral supernatant was
spinoculated at \SI{2000}{\gforce} for \SI{2}{\hour} at \SI{4}{\degreeCelsius}.
T cells were activated in 96 well plates using beads or \glspl{dms} for
\SI{24}{\hour}, and then cells and beads/\glspl{dms} were transferred onto
lentiviral vector coated plates and incubated for another \SI{24}{\hour}. Cells
and beads/\glspl{dms} were removed from the retronectin plates using vigorous
pipetting and transferred to another 96 well plate wherein expansion continued.
% METHOD fill in missing product numbers
\gls{bcma} \gls{car} lentiviral vector was synthesized in house as
follows\footnote{lentiviral synthesis was performed by Ritika Jain in our
laboratory and included here for reference}. \SI{10}{\ng} of
\anti{\gls{bcma}}-CD8-CD137-CD3$\upzeta$ plasmid (generously provided by Jim
Kochenderfer at the NIH)\cite{Lam2020} was added to \SI{50}{\ul}
\product{DH5$\upalpha$ cells}{\thermo}{18265017} and incubated for
\SI{30}{\minute} on ice. The cell mixture was then heat-shocked at
\SI{42}{\degreeCelsius} for \SI{20}{\minute} before being placed on ice for
another \SI{2}{\minute}. \SI{950}{\ul} \product{LB Broth}{TODO}{TODO} was added
to the cells which were then centrifuged for \SI{1}{\hour} at \SI{225}{\rpm}.
\SI{20}{\ul} of the cell mixture was then spread over selection plates and
incubated overnight at \SI{37}{\degreeCelsius}. Colonies were selected the
following day and incubated in \product{LB Broth}{TODO}{TODO} with
\product{ampicillin}{\sigald{}}{A9518-5G} at \SI{37}{\degreeCelsius} for
\SIrange{12}{16}{\hour} prior to using the \product{miniprep kit}{Qiagen}{27104}
as per the manufacturer's instructions to isolate the plasmid DNA. Transfer
plasmid along with \product{pMDLg/pRRE}{Addgene}{12251},
\product{pRSV-Rev}{Addgene}{12253}, and \product{pMD2.G}{Addgene}{12259}
(generously provided by the Sloan lab at Emory University) in
\product{Opti-Mem}{\thermo}{31-985-070} with \product{lipfectamine
2000}{\thermo}{11668019} were added dropwise to HEK 293T cells and incubated
for \SI{6}{\hour}, after which all media was replaced with fresh fresh media.
After \SI{24}{\hour} and \SI{48}{\hour}, supernatent was collected, pooled, and
concentrated using a \product{Lenti-X concentrator}{Takara}{631231} prior to
storing at \SI{-80}{\degreeCelsius}.
\subsection{sulfo-NHS-biotin hydrolysis quantification}
@ -3375,8 +3404,8 @@ interest using \glspl{mab}.
% METHOD The concentration for the surface marker cleavage experiment was much
% higher, if that matters
\glspl{dms} were digested in active T cell cultures via addition of sterile
\product{\gls{colb}}{Sigma}{11088807001} or
\product{\gls{cold}}{Sigma}{11088858001}. Collagenase was dissolved in
\product{\gls{colb}}{\sigald}{11088807001} or
\product{\gls{cold}}{\sigald}{11088858001}. Collagenase was dissolved in
\product{\gls{hbss}}{Gibco}{14025-076} or
\product{TexMACS}{\miltenyi}{170-076-307} at approximately \SI{100}{\ug\per\ml}.
This solution was added to T cell cultures at a 1:1 ratio in place of plain
@ -3405,8 +3434,8 @@ calculation neighborhood size of 5 and local density approximation factor of
\subsection{integrin blocking experiments}
To block \gls{a2b1} and \gls{a2b2}, active T cell cultures with \gls{dms} were
supplemented with \product{\anti{\gls{a2b1}}}{Sigma}{MAB1973Z} and
\product{\anti{\gls{a2b2}}}{Sigma}{MAB1950Z} (both \gls{leaf}) at indicated
supplemented with \product{\anti{\gls{a2b1}}}{\sigald}{MAB1973Z} and
\product{\anti{\gls{a2b2}}}{\sigald}{MAB1950Z} (both \gls{leaf}) at indicated
concentrations and timepoints. T cells were grown as described in
\cref{sec:tcellculture}.