ADD bcma car section
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@ -2200,7 +2200,7 @@ CONCLUSIONS: We developed a simplified, semi-closed system for the initial selec
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country = {United States},
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country = {United States},
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issn-linking = {1538-4047},
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issn-linking = {1538-4047},
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issue = {6},
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issue = {6},
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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},
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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},
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nlm-id = {101137842},
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nlm-id = {101137842},
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owner = {NLM},
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owner = {NLM},
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pii = {557},
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pii = {557},
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@ -2463,6 +2463,18 @@ CONCLUSIONS: We developed a simplified, semi-closed system for the initial selec
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url = {https://www.ebook.de/de/product/8324352/wu\_hamada\_experiments\_2e.html},
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url = {https://www.ebook.de/de/product/8324352/wu\_hamada\_experiments\_2e.html},
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}
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}
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@Article{Lam2020,
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author = {Norris Lam and Nathan D. Trinklein and Benjamin Buelow and George H. Patterson and Namrata Ojha and James N. Kochenderfer},
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journal = {Nature Communications},
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title = {Anti-{BCMA} chimeric antigen receptors with fully human heavy-chain-only antigen recognition domains},
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year = {2020},
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month = {jan},
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number = {1},
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volume = {11},
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doi = {10.1038/s41467-019-14119-9},
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publisher = {Springer Science and Business Media {LLC}},
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}
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@Comment{jabref-meta: databaseType:bibtex;}
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@Comment{jabref-meta: databaseType:bibtex;}
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@Comment{jabref-meta: grouping:
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@Comment{jabref-meta: grouping:
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@ -162,6 +162,7 @@
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\newacronym{talen}{TALEN}{transcription activator-like effector nuclease}
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\newacronym{talen}{TALEN}{transcription activator-like effector nuclease}
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\newacronym{qbd}{QbD}{quality-by-design}
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\newacronym{qbd}{QbD}{quality-by-design}
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\newacronym{aws}{AWS}{amazon web services}
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\newacronym{aws}{AWS}{amazon web services}
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\newacronym{qpcr}{qPCR}{quantitative polymerase chain reaction}
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%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%
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%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%
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% SI units for uber nerds
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% SI units for uber nerds
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@ -243,6 +244,7 @@
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\newcommand{\catnum}[2]{(#1, #2)}
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\newcommand{\catnum}[2]{(#1, #2)}
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\newcommand{\product}[3]{#1 \catnum{#2}{#3}}
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\newcommand{\product}[3]{#1 \catnum{#2}{#3}}
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\newcommand{\thermo}{Thermo Fisher}
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\newcommand{\thermo}{Thermo Fisher}
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\newcommand{\sigald}{Sigma Aldrich}
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\newcommand{\miltenyi}{Miltenyi Biotech}
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\newcommand{\miltenyi}{Miltenyi Biotech}
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\newcommand{\bl}{Biolegend}
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\newcommand{\bl}{Biolegend}
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\newcommand{\bd}{Becton Dickenson}
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\newcommand{\bd}{Becton Dickenson}
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@ -743,7 +745,7 @@ material used for tissue culture flasks and dishes in general) with no
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modification (SoloHill Plastic, Nunc Biosilon), with a cationic surface charge
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modification (SoloHill Plastic, Nunc Biosilon), with a cationic surface charge
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(SoloHill Hillex) or coated with an \gls{ecm} protein such as collagen (SoloHill
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(SoloHill Hillex) or coated with an \gls{ecm} protein such as collagen (SoloHill
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Fact III). Other base materials have been used such as dextran (GE Healthcare
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Fact III). Other base materials have been used such as dextran (GE Healthcare
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Cytodex), cellulose (GE Healthcare Cytopore), and glass (Sigma Aldrich G2767),
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Cytodex), cellulose (GE Healthcare Cytopore), and glass (\sigald{} G2767),
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all with none or similar surface modifications. Additionally, some microcarriers
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all with none or similar surface modifications. Additionally, some microcarriers
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such as \gls{cus} and \gls{cug} are composed entirely out of protein (in these
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such as \gls{cus} and \gls{cug} are composed entirely out of protein (in these
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cases, porcine collagen) which also allows the microcarriers to be enzymatically
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cases, porcine collagen) which also allows the microcarriers to be enzymatically
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@ -1281,7 +1283,7 @@ two washes were done and the agitation time was \SI{30}{\minute}. Biotinylated
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\glspl{mab} against human CD3 \catnum{\bl}{317320} and CD28 \catnum{\bl}{302904}
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\glspl{mab} against human CD3 \catnum{\bl}{317320} and CD28 \catnum{\bl}{302904}
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were combined in a 1:1 mass ratio and added to the carriers at
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were combined in a 1:1 mass ratio and added to the carriers at
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\SI{0.2}{\ug\of{\ab}\per\mg\of{\dms}}. Along with the \glspl{mab}, sterile
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\SI{0.2}{\ug\of{\ab}\per\mg\of{\dms}}. Along with the \glspl{mab}, sterile
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\product{\gls{bsa}}{Sigma}{A9576} was added to a final concentration of
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\product{\gls{bsa}}{\sigald}{A9576} was added to a final concentration of
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\SI{2}{\percent} in order to prevent non-specific binding of the antibodies to
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\SI{2}{\percent} in order to prevent non-specific binding of the antibodies to
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the reaction tubes. \glspl{mab} were allowed to bind to the carriers for
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the reaction tubes. \glspl{mab} were allowed to bind to the carriers for
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\SI{60}{\minute} with \SI{700}{\rpm} agitation. After binding, supernatants were
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\SI{60}{\minute} with \SI{700}{\rpm} agitation. After binding, supernatants were
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@ -1299,12 +1301,12 @@ was then manually counted to obtain a concentration. Surface area for
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\subsection{dms quality control assays}
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\subsection{dms quality control assays}
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Biotin was quantified using the \product{\gls{haba} assay}{Sigma}{H2153-1VL}. In
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Biotin was quantified using the \product{\gls{haba} assay}{\sigald}{H2153-1VL}.
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the case of quantifying \gls{snb} prior to adding it to the microcarriers, the
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In the case of quantifying \gls{snb} prior to adding it to the microcarriers,
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sample volume was quenched in a 1:1 volumetric ratio with \SI{1}{\molar} NaOH
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the sample volume was quenched in a 1:1 volumetric ratio with \SI{1}{\molar}
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and allowed to react for \SI{1}{\minute} in order to prevent the reactive ester
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NaOH and allowed to react for \SI{1}{\minute} in order to prevent the reactive
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linkages from binding to the avidin proteins in the \gls{haba}/avidin premix.
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ester linkages from binding to the avidin proteins in the \gls{haba}/avidin
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All quantifications of \gls{haba} were performed on an Eppendorf D30
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premix. All quantifications of \gls{haba} were performed on an Eppendorf D30
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Spectrophotometer using \product{\SI{70}{\ul} cuvettes}{BrandTech}{759200}. The
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Spectrophotometer using \product{\SI{70}{\ul} cuvettes}{BrandTech}{759200}. The
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extinction coefficient at \SI{500}{\nm} for \gls{haba}/avidin was assumed to be
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extinction coefficient at \SI{500}{\nm} for \gls{haba}/avidin was assumed to be
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\SI{34000}{\per\cm\per\molar}.
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\SI{34000}{\per\cm\per\molar}.
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@ -1457,25 +1459,52 @@ Briefly, cells were washed once and stained with \product{biotinylated
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BD Accuri. Readout was percent \gls{pe}+ cells as compared to secondary controls
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BD Accuri. Readout was percent \gls{pe}+ cells as compared to secondary controls
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(\gls{pe}-\gls{stp} with no \gls{ptnl}).
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(\gls{pe}-\gls{stp} with no \gls{ptnl}).
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% TODO add BCMA-CAR stuff
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\subsection{car plasmid and lentiviral transduction}
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\subsection{car plasmid and lentiviral transduction}
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The anti-CD19-CD8-CD137-CD3z \gls{car} with the EF1$\upalpha$
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The anti-CD19-CD8-CD137-CD3$\upzeta$ \gls{car} with the EF1$\upalpha$
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promotor\cite{Milone2009} was synthesized (Aldevron) and subcloned into a
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promotor\cite{Milone2009} was synthesized (Aldevron) and subcloned into a
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\product{FUGW}{Addgene}{14883} kindly provided by the Emory Viral Vector Core.
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\product{FUGW}{Addgene}{14883} kindly provided by the Emory Viral Vector Core.
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Lentiviral vectors were synthesized by the Emory Viral Vector Core or the
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Lentiviral vectors were synthesized by the Emory Viral Vector Core or the
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Cincinnati Children's Hospital Medical Center Viral Vector Core. To transduce
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Cincinnati Children's Hospital Medical Center Viral Vector Core. RNA titer was
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primary human T cells, \product{retronectin}{Takara}{T100A} was coated onto
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calculated using a \product{Lenti-X \gls{qpcr} titer kit}{Takara}{631235}. To
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non-TC treated 96 well plates and used to immobilize lentiviral vector particles
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transduce primary human T cells, \product{retronectin}{Takara}{T100A} was coated
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according to the manufacturer's instructions. Briefly, retronectin solution was
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onto non-TC treated 96 well plates and used to immobilize lentiviral vector
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adsorbed overnight at \SI{4}{\degreeCelsius} and blocked the next day using
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particles according to the manufacturer's instructions. Briefly, retronectin
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\gls{bsa}. Prior to transduction, lentiviral supernatant was spinoculated at
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solution was adsorbed overnight at \SI{4}{\degreeCelsius} and blocked the next
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\SI{2000}{\gforce} for \SI{2}{\hour} at \SI{4}{\degreeCelsius}. T cells were
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day using \gls{bsa}. Prior to transduction, lentiviral supernatant was
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activated in 96 well plates using beads or \glspl{dms} for \SI{24}{\hour}, and
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spinoculated at \SI{2000}{\gforce} for \SI{2}{\hour} at \SI{4}{\degreeCelsius}.
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then cells and beads/\glspl{dms} were transferred onto lentiviral vector coated
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T cells were activated in 96 well plates using beads or \glspl{dms} for
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plates and incubated for another \SI{24}{\hour}. Cells and beads/\glspl{dms}
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\SI{24}{\hour}, and then cells and beads/\glspl{dms} were transferred onto
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were removed from the retronectin plates using vigorous pipetting and
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lentiviral vector coated plates and incubated for another \SI{24}{\hour}. Cells
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transferred to another 96 well plate wherein expansion continued.
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and beads/\glspl{dms} were removed from the retronectin plates using vigorous
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pipetting and transferred to another 96 well plate wherein expansion continued.
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% METHOD fill in missing product numbers
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\gls{bcma} \gls{car} lentiviral vector was synthesized in house as
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follows\footnote{lentiviral synthesis was performed by Ritika Jain in our
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laboratory and included here for reference}. \SI{10}{\ng} of
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\anti{\gls{bcma}}-CD8-CD137-CD3$\upzeta$ plasmid (generously provided by Jim
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Kochenderfer at the NIH)\cite{Lam2020} was added to \SI{50}{\ul}
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\product{DH5$\upalpha$ cells}{\thermo}{18265017} and incubated for
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\SI{30}{\minute} on ice. The cell mixture was then heat-shocked at
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\SI{42}{\degreeCelsius} for \SI{20}{\minute} before being placed on ice for
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another \SI{2}{\minute}. \SI{950}{\ul} \product{LB Broth}{TODO}{TODO} was added
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to the cells which were then centrifuged for \SI{1}{\hour} at \SI{225}{\rpm}.
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\SI{20}{\ul} of the cell mixture was then spread over selection plates and
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incubated overnight at \SI{37}{\degreeCelsius}. Colonies were selected the
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following day and incubated in \product{LB Broth}{TODO}{TODO} with
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\product{ampicillin}{\sigald{}}{A9518-5G} at \SI{37}{\degreeCelsius} for
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\SIrange{12}{16}{\hour} prior to using the \product{miniprep kit}{Qiagen}{27104}
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as per the manufacturer's instructions to isolate the plasmid DNA. Transfer
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plasmid along with \product{pMDLg/pRRE}{Addgene}{12251},
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\product{pRSV-Rev}{Addgene}{12253}, and \product{pMD2.G}{Addgene}{12259}
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(generously provided by the Sloan lab at Emory University) in
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\product{Opti-Mem}{\thermo}{31-985-070} with \product{lipfectamine
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2000}{\thermo}{11668019} were added dropwise to HEK 293T cells and incubated
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for \SI{6}{\hour}, after which all media was replaced with fresh fresh media.
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After \SI{24}{\hour} and \SI{48}{\hour}, supernatent was collected, pooled, and
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concentrated using a \product{Lenti-X concentrator}{Takara}{631231} prior to
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storing at \SI{-80}{\degreeCelsius}.
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\subsection{sulfo-NHS-biotin hydrolysis quantification}
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\subsection{sulfo-NHS-biotin hydrolysis quantification}
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@ -3375,8 +3404,8 @@ interest using \glspl{mab}.
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% METHOD The concentration for the surface marker cleavage experiment was much
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% METHOD The concentration for the surface marker cleavage experiment was much
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% higher, if that matters
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% higher, if that matters
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\glspl{dms} were digested in active T cell cultures via addition of sterile
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\glspl{dms} were digested in active T cell cultures via addition of sterile
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\product{\gls{colb}}{Sigma}{11088807001} or
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\product{\gls{colb}}{\sigald}{11088807001} or
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\product{\gls{cold}}{Sigma}{11088858001}. Collagenase was dissolved in
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\product{\gls{cold}}{\sigald}{11088858001}. Collagenase was dissolved in
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\product{\gls{hbss}}{Gibco}{14025-076} or
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\product{\gls{hbss}}{Gibco}{14025-076} or
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\product{TexMACS}{\miltenyi}{170-076-307} at approximately \SI{100}{\ug\per\ml}.
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\product{TexMACS}{\miltenyi}{170-076-307} at approximately \SI{100}{\ug\per\ml}.
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This solution was added to T cell cultures at a 1:1 ratio in place of plain
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This solution was added to T cell cultures at a 1:1 ratio in place of plain
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@ -3405,8 +3434,8 @@ calculation neighborhood size of 5 and local density approximation factor of
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\subsection{integrin blocking experiments}
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\subsection{integrin blocking experiments}
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To block \gls{a2b1} and \gls{a2b2}, active T cell cultures with \gls{dms} were
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To block \gls{a2b1} and \gls{a2b2}, active T cell cultures with \gls{dms} were
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supplemented with \product{\anti{\gls{a2b1}}}{Sigma}{MAB1973Z} and
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supplemented with \product{\anti{\gls{a2b1}}}{\sigald}{MAB1973Z} and
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\product{\anti{\gls{a2b2}}}{Sigma}{MAB1950Z} (both \gls{leaf}) at indicated
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\product{\anti{\gls{a2b2}}}{\sigald}{MAB1950Z} (both \gls{leaf}) at indicated
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concentrations and timepoints. T cells were grown as described in
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concentrations and timepoints. T cells were grown as described in
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\cref{sec:tcellculture}.
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\cref{sec:tcellculture}.
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