FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2786393802> ?p ?o ?g. }

• W2786393802 endingPage "1915" @default.
• W2786393802 startingPage "1915" @default.
• W2786393802 abstract "Chimeric antigen receptor (CAR)-based cellular therapy is a revolutionary approach to treat cancer as witnessed by recent success in clinical trials for various hematological malignancies. Currently, flow cytometry based detection of fluorochrome-tagged antibodies or proteinL that binds to the Extra-Cellular Domain (ECD) of CAR molecule are the widely used methods for the detection of CARexpression. Here, we have developed a novel luciferase based assay for detecting the expression of CAR. Our assay is accurate, highly sensitive (10-5), and has a broad linearity by taking advantage of the extreme brightness of recently discovered marine luciferases (Gluc/Nluc/Tluc16/Mluc/Loluc/Paluc/Htluc). The assay is based on recombinant fusion protein technology by fusing the ECD of a CAR target in frame with one of the marine luciferases (for detection) along with several small peptide tags -Flag/ Strep -tag II/AcV5/His (for isolation). Initially, a fusion construct was made by cloning the ECD of CD19 fused in frame with Nluc. The fusion protein was produced using 293FT cells, and tested by a simple binding assay that involved 45 minutes incubation at 4oC followed by washing and detection of luminescence. More than 103 fold increases in luminescence was observed between FMC63-CARtransduced-T/NK cells and uninfected cells or a non-specific CAR transduced cells. Essentially, identical results were obtained by replacing Nluc with other marine luciferases or by using cells transduced with five distinct CARs targeting CD19. Similar strategy was successfully applied for the specific detection of CARs targeting CD20, CD30, CD33, CD123, CD138, BCMA, and SLAM7. We also show that the small peptide tags in the fusion protein can be used for the specific isolation of CAR+ve cells using anti-tag antibodies by FACS. Additionally, we purified ECD-fusion proteins for CD19 and CD33 using Strep -Tactin protein purification columns. Purified fusion proteins were fully active, as observed by successful and specific binding to respective CAR-T/NK cells. Furthermore, direct conjugation of purified ECD-fusion proteins with fluorochromes resulted in a single-step detection and/or isolation of CAR+vecells. In another embodiment, an antigen positive cell can be detected by fusing the single-chain fragment variable (scFv) of its specific monoclonal antibody with one of the marine luciferases. A fusion construct was made by cloning scFv of a CD19 antibody clone (FMC63) fused in frame with Nluc, followed by the fusion protein production and binding assay. Again, a 103 fold increase in luminescence was observed between CD19+ve cell lines (Raji, Nalm6, and BV173) and CD19-ve cell lines (HL60 or Raji-CD19-/- by Crispr-Cas9). Similar results were obtained using other marine luciferases in place of Nluc or by replacing FMC63 with five distinct scFv targeting CD19. Next, we made fusion proteins using scFv of antibody clones targeting CD20, CD30, CD33, CD123, CD138, BCMA, and SLAM7 and tested by binding assay using a panel of cell lines comprising 8 hematological malignancies. Strikingly, all the scFv fusion proteins bound strictly to their respective antigen+ve cell lines. Until now, only a limited number of targets have been pursued using CAR technology, which can be attributed to the lack of a High Throughput Screening (HTS)-based approach to find novel targets. To check the compatibility of our scFv-luciferase fusion protein technology as a HTS approach to discover novel CAR targets, a library of 78 distinct scFv-Nluc constructs were made, followed by the production of fusion proteins. Binding assays were performed in 96-well plates using Raji, BV173, MM1S, and U266 cell lines. Fusion proteins which are supposed to bind their respective targets were readily identified by the assay - Raji (CD19 and CD20 targeting scFv); BV173 (CD19 and CD33 targeting scFv); MM1S and U266 (CD138, BCMA, and SLAM7 targeting scFv). Most importantly, few novel CAR targets were identified thereby opening up the possibility of applying this screening approach to patient tumor cells, that may result in precision medicine model in cellular-therapy space by tailoring CAR therapy for individual patients. Finally, we demonstrate the utility of scFv-luciferase fusion protein technology in accurately detecting the serial engraftment of B-ALL and AML-patient derived xenografts in NSG mice using scFv of antibodies targeting CD19 and CD33, respectively. Disclosures Chaudhary: University of Southern California: Patents & Royalties: P.M.C. have filed patent application related to the technology described in this manuscript." @default.
• W2786393802 created "2018-02-23" @default.
• W2786393802 creator A5003425745 @default.
• W2786393802 creator A5003812373 @default.
• W2786393802 creator A5005892609 @default.
• W2786393802 creator A5008647842 @default.
• W2786393802 creator A5010550624 @default.
• W2786393802 creator A5011579156 @default.
• W2786393802 creator A5017979600 @default.
• W2786393802 creator A5020343857 @default.
• W2786393802 creator A5022581092 @default.
• W2786393802 creator A5027222530 @default.
• W2786393802 creator A5028770749 @default.
• W2786393802 creator A5031023632 @default.
• W2786393802 creator A5031955495 @default.
• W2786393802 creator A5038450006 @default.
• W2786393802 creator A5038811365 @default.
• W2786393802 creator A5048372827 @default.
• W2786393802 creator A5048578171 @default.
• W2786393802 creator A5053450219 @default.
• W2786393802 creator A5054056420 @default.
• W2786393802 creator A5061334100 @default.
• W2786393802 creator A5061866803 @default.
• W2786393802 creator A5063244757 @default.
• W2786393802 creator A5069986166 @default.
• W2786393802 creator A5071957689 @default.
• W2786393802 creator A5090614479 @default.
• W2786393802 creator A5090703420 @default.
• W2786393802 creator A5091277968 @default.
• W2786393802 date "2017-12-07" @default.
• W2786393802 modified "2023-09-28" @default.
• W2786393802 title "A Novel Method for Highly Sensitive Detection and Isolation of Chimeric Antigen Receptor and Cancer Associated Antigen-Expressing Cells" @default.
• W2786393802 doi "https://doi.org/10.1182/blood.v130.suppl_1.1915.1915" @default.
• W2786393802 hasPublicationYear "2017" @default.
• W2786393802 type Work @default.
• W2786393802 sameAs 2786393802 @default.
• W2786393802 citedByCount "0" @default.
• W2786393802 crossrefType "journal-article" @default.
• W2786393802 hasAuthorship W2786393802A5003425745 @default.
• W2786393802 hasAuthorship W2786393802A5003812373 @default.
• W2786393802 hasAuthorship W2786393802A5005892609 @default.
• W2786393802 hasAuthorship W2786393802A5008647842 @default.
• W2786393802 hasAuthorship W2786393802A5010550624 @default.
• W2786393802 hasAuthorship W2786393802A5011579156 @default.
• W2786393802 hasAuthorship W2786393802A5017979600 @default.
• W2786393802 hasAuthorship W2786393802A5020343857 @default.
• W2786393802 hasAuthorship W2786393802A5022581092 @default.
• W2786393802 hasAuthorship W2786393802A5027222530 @default.
• W2786393802 hasAuthorship W2786393802A5028770749 @default.
• W2786393802 hasAuthorship W2786393802A5031023632 @default.
• W2786393802 hasAuthorship W2786393802A5031955495 @default.
• W2786393802 hasAuthorship W2786393802A5038450006 @default.
• W2786393802 hasAuthorship W2786393802A5038811365 @default.
• W2786393802 hasAuthorship W2786393802A5048372827 @default.
• W2786393802 hasAuthorship W2786393802A5048578171 @default.
• W2786393802 hasAuthorship W2786393802A5053450219 @default.
• W2786393802 hasAuthorship W2786393802A5054056420 @default.
• W2786393802 hasAuthorship W2786393802A5061334100 @default.
• W2786393802 hasAuthorship W2786393802A5061866803 @default.
• W2786393802 hasAuthorship W2786393802A5063244757 @default.
• W2786393802 hasAuthorship W2786393802A5069986166 @default.
• W2786393802 hasAuthorship W2786393802A5071957689 @default.
• W2786393802 hasAuthorship W2786393802A5090614479 @default.
• W2786393802 hasAuthorship W2786393802A5090703420 @default.
• W2786393802 hasAuthorship W2786393802A5091277968 @default.
• W2786393802 hasConcept C104317684 @default.
• W2786393802 hasConcept C111335760 @default.
• W2786393802 hasConcept C121608353 @default.
• W2786393802 hasConcept C123894998 @default.
• W2786393802 hasConcept C147483822 @default.
• W2786393802 hasConcept C153911025 @default.
• W2786393802 hasConcept C185592680 @default.
• W2786393802 hasConcept C203014093 @default.
• W2786393802 hasConcept C2777701055 @default.
• W2786393802 hasConcept C2778957590 @default.
• W2786393802 hasConcept C2909536089 @default.
• W2786393802 hasConcept C3875195 @default.
• W2786393802 hasConcept C40767141 @default.
• W2786393802 hasConcept C54009773 @default.
• W2786393802 hasConcept C54355233 @default.
• W2786393802 hasConcept C553184892 @default.
• W2786393802 hasConcept C55493867 @default.
• W2786393802 hasConcept C81885089 @default.
• W2786393802 hasConcept C86803240 @default.
• W2786393802 hasConceptScore W2786393802C104317684 @default.
• W2786393802 hasConceptScore W2786393802C111335760 @default.
• W2786393802 hasConceptScore W2786393802C121608353 @default.
• W2786393802 hasConceptScore W2786393802C123894998 @default.
• W2786393802 hasConceptScore W2786393802C147483822 @default.
• W2786393802 hasConceptScore W2786393802C153911025 @default.
• W2786393802 hasConceptScore W2786393802C185592680 @default.
• W2786393802 hasConceptScore W2786393802C203014093 @default.
• W2786393802 hasConceptScore W2786393802C2777701055 @default.
• W2786393802 hasConceptScore W2786393802C2778957590 @default.
• W2786393802 hasConceptScore W2786393802C2909536089 @default.
• W2786393802 hasConceptScore W2786393802C3875195 @default.
• W2786393802 hasConceptScore W2786393802C40767141 @default.
• W2786393802 hasConceptScore W2786393802C54009773 @default.

• next