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Engineered immune effector cells and use thereof

A technology of hematopoietic cells and cells, applied in the field of engineered immune effector cells and their uses, can solve problems such as unmet improvement

Pending Publication Date: 2020-11-17
FATE THERAPEUTICS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, using these immune cells for adoptive cell therapy remains challenging and there is an unmet need for improvement

Method used

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  • Engineered immune effector cells and use thereof
  • Engineered immune effector cells and use thereof
  • Engineered immune effector cells and use thereof

Examples

Experimental program
Comparison scheme
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example

[0346] The following examples are provided for illustration, not limitation.

[0347] Example 1 - Materials and methods

[0348] In order to efficiently select and test suicide systems under the control of different promoters combined with different safe harbor locus integration strategies, the applicant's proprietary hiPSC platform, which is capable of single cell passage and high-throughput 96-based Flow cytometric sorting of well plates to derive clonal hiPSCs with single or multiple gene regulation.

[0349] Maintenance of hiPSCs in small molecule culture: hiPSCs are conventionally passaged as single cells after the culture confluency reaches 75%-90%. For single cell dissociation, hiPSCs were washed once with PBS (Mediatech) and treated with Accutase (Millipore) for 3-5 min at 37°C, followed by pipetting to ensure single cell dissociation. The single cell suspension was then mixed with an equal volume of conventional medium, centrifuged at 225 xg for 4 minutes, resuspend...

example 2

[0352] Example 2 - CD38 knockout in iPSCs using CRISPR / Cas9-mediated genome editing

[0353] Alt- S.p.Cas9 D10A Nickase 3NLS (100μg) and Alt- CRISPR-Cas9 tracrRNA was purchased from IDT (Coralville, Iowa) and used for iPSC targeted editing. For biallelic knockout of CD38 in iPSCs using Cas9 nickase, the screened and identified targeting sequence pairs designed for gNA (i.e., gD / RNA or guide polynucleotide) are listed in Table 3 (1A and 1B, 2A and 2B, 3A and 3B):

[0354] Table 3: Targeting sequences specific to the CD38 locus for CRISPR / Cas9 genome editing:

[0355] Exon / Chr# targeting sequence PAM cleavage site SEQ ID NO: CD38-gNA-1A 1 / 4 TTGACGCATCGCGCCAGGA CGG 15,778,604 1 CD38-gNA-1B 1 / 4 ATTCATCCTGAGATGAGGT GGG 15,778,646 2 CD38-gNA-2A 1 / 4 ACTGACGCCAAGACAGAGT TGG 15,778,485 3 CD38-gNA-2B 1 / 4 CTGGTCCTGATCCTCGTCG TGG 15,778,520 4 CD38-gNA-3A 1 / 4 TCCTAGAGAGCCGGCAGCA GGG 15,778,459 5 CD38-...

example 3-CD3

[0357] Example 3-CD38 - / - Validation of iPSCs and derived cells

[0358] CD38 is known to be expressed at specific cell stages and plays a key role in effector cells. During hematopoiesis, CD38 in CD34 + Expressed on stem cells and lineage specialized progenitors of lymphoid, erythroid and myeloid, and also during the final stages of maturation of effector cells such as T cells and NK cells. Thus, prior to this application, it was unknown and worrisome whether iPSCs comprising a CD38 knockout would develop normally when subjected to directed differentiation conditions, and whether the resulting effector cells would function as CD38 expression profiles and functions. effect. CD38-knockout iPSCs containing biallelic knockout of CD38 surprisingly maintained their ability to differentiate into derivative cells. In one of the illustrations, three engineered iPSC clones gene-edited to have a biallelic disruption of the CD38 gene and hnCD16 were differentiated into derived NK cel...

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Abstract

Provided are methods and compositions for obtaining functionally enhanced derivative effector cells obtained from directed differentiation of genomically engineered iPSCs. The derivative cells provided herein have stable and functional genome editing that delivers improved or enhanced therapeutic effects. Also provided are therapeutic compositions and the used thereof comprising the functionally enhanced derivative effector cells alone, or with antibodies or checkpoint inhibitors in combination therapies.

Description

[0001] related application [0002] This application claims U.S. Provisional Application Serial No. 62 / 649,781 filed March 29, 2018, U.S. Provisional Application Serial No. 62 / 774,052 filed November 30, 2018, and International Application No. PCT / Priority to US 18 / 67289, the disclosure of which is incorporated herein by reference in its entirety. [0003] References to Sequence Listings Submitted Electronically [0004] This application incorporates by reference the computer-readable format (CRF) of the Sequence Listing filed with this application in ASCII text entitled 13601-204-228_SEQ_LISTING.txt, created on March 28, 2018, and The size is 45,867 bytes. technical field [0005] The present disclosure is broadly relevant to the field of off-the-shelf immune cell products. More specifically, the present disclosure relates to strategies for developing multifunctional effector cells capable of delivering therapeutically relevant properties in vivo. The cell products dev...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N5/0789C12N5/074C07K14/54C07K14/735C07K14/715
CPCC12N9/2497C07K14/7155C07K14/5443C07K14/70535C12N15/90C07K2319/00C12N5/0646C12N2510/00C12N2506/45C12N2533/90C12N2533/52C12N2310/20C12N15/1138A61K39/4613A61K39/4611A61K39/4631A61K39/464426C12N15/85C12N5/0647C12N5/0696C12N2840/002C12N5/0636C07K14/70596C12N5/10C12N15/907
Inventor B·瓦拉梅尔R·比乔戴尔J·古德里奇T·T·李
Owner FATE THERAPEUTICS
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