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Modulating expression of polypeptides via new gene switch expression systems

A gene switch and heterologous gene technology, applied in the field of regulating polypeptide expression through a new gene switch expression system, can solve problems such as insufficient efficacy and no safety issues

Pending Publication Date: 2019-11-15
INTREXON CORP (US)
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

While this innovative technology is promising, administering modified T cells to tumor-bearing individuals is not without safety concerns, such as tumor lysis and cytokine release syndrome
Additionally, expression of CAR or TCR alone may not be sufficient to achieve efficacy and additional expression of cytokines such as IL-2, IL-12, IL-15 or IL-21 may be required to increase the efficacy of such treatments
However, this may cause additional security issues

Method used

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  • Modulating expression of polypeptides via new gene switch expression systems
  • Modulating expression of polypeptides via new gene switch expression systems
  • Modulating expression of polypeptides via new gene switch expression systems

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0499] Embodiment 1.Nucleofection of PBMC by SB gene switch system

[0500] Various DNA plasmids expressing the SB transposon system, namely SB11, membrane-bound IL-15 (mbIL-15) and chimeric antigen receptor (CAR), were transfected into peripheral blood mononuclear cells (PBMCs) by nucleofection. ), to redirect T cell specificity ( figure 1 ).

[0501] On day 0, 20 million PBMCs were resuspended in 100 μl of Amaxa Human T cell Nucleofector solution (catalog number VPA-1002; Lonza, Basel, Switzerland) mixed with a total of 15 μg of transposons and 5 μg of transposase (SB11) , and perform electroporation.

[0502]Cells were counted the next day (Day 1) and CAR expression was measured by flow cytometry. CAR T cells were stimulated at a 1:1 ratio with γ-irradiated (100 Gy) or mitomycin C-treated AaPC. The AaPC ​​cells used were K562-AaPC expressing CD19 antigen. Cultures were supplemented with IL-21 (30 ng / ml) for the first round of stimulation only, followed by recombinant ...

Embodiment 2

[0503] Example 2. Flow cytometry analysis of ON-OFF SB gene switch system

[0504] CD19-specific T cells were generated from PB- or UCB-derived monocytes (MNCs), in which CAR was introduced using SB transposition, followed by addition of AaPCs, to numerically expand T cells in a CAR-dependent manner.

[0505] For flow cytometry analysis to assess expression of various markers and genes of interest, cells were gently resuspended and cell number and viability were measured with a Countess instrument using trypan blue exclusion. Cell diameter size was also recorded. Harvest 5×10 per sample at 330×g for 4 min at 10° C. 5 cells for antibody staining. Harvested cells were incubated with 10% human AB serum in HBSS for at least 15 min on ice. Antibody cocktail containing fluorescently conjugated antibodies in HBSS + 0.1% BSA + 2mM EDTA including one or more specific for CD4 (clone RPA-T4), CD8 (clone SK1), CD3 (clone UCHT1), CD56, CD19 CAR (anti-idiotypic antibody), IL-15 and IL-1...

Embodiment 3

[0508] Example 3. Survival experiment

[0509] Multiple Sleeping Beauty transposon DNA plasmids (design 9, Fig. 2) transfected with the plasmid DNA encoding Sleeping Beauty transposase SB11 into peripheral blood mononuclear cells (PBMC) ( figure 1 ). On day 0, 20 million PBMCs were resuspended in 100 μl Amaxa Human T cell Nucleofector solution (catalog number VPA-1002; Lonza, Basel, Switzerland) mixed with a total of 15 μg transposon and 5 μg transposase, and nucleated. transfected into cells. Cells were counted the next day (Day 1) and CAR expression was measured using flow cytometry. Cells were stimulated at a 1:1 ratio with γ-irradiated (100 Gy) or mitomycin C-treated K562-based AaPC ​​expressing CD19 on their surface. Cultures were supplemented with IL-21 (30 ng / ml) for the first round of stimulation only, followed by recombinant human IL-2 (50 IU / ml) and IL-21 (30 ng / ml) (Pepro Tech) for the remainder of the stimulation. T cell cultures were phenotyped at the end of...

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Abstract

Disclosed herein are polynucleotides encoding ligand-inducible gene switch polypeptides, and systems comprising gene switch polypeptides for modulating the expression of a heterologous gene and an interleukin in a host cell. The compositions, methods and systems described herein facilitate ligand dependent expression of polypeptides including but not limited to cytokines and antigen binding polypeptides.

Description

[0001] Cross References to Related Applications [0002] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 444,775, filed January 10, 2017, and U.S. Provisional Patent Application No. 62 / 464,958, filed February 28, 2017, which are hereby incorporated by reference in their entirety . [0003] References to Sequence Listings [0004] This application contains a Sequence Listing, which has been filed electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy was created on January 9, 2018, is named 50471_706_601_SL.txt, and is 421,163 bytes in size. [0005] Incorporate by reference [0006] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. Background technique [0007...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12N15/63G01N33/68
CPCC12N15/1055C12N15/63G01N33/6845C07K2319/03C07K14/7051A61K39/4611A61K2239/23A61K39/4631A61K2239/38A61K39/464412A61K39/464404A61K2239/47A61K39/4635C12N15/635C12N15/85C12N2800/90C12N15/907C12N2830/002C07K14/5443C07K2319/80C12N2840/203
Inventor 鲁图尔·R·沙阿托马斯·D·里德谢丽尔·G·博林格
Owner INTREXON CORP (US)
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