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Methods and compositions for genetically modifying and expanding lymphocytes and regulating the activity thereof

A technology of lymphoid tissue and cells, applied in the field of immunology

Pending Publication Date: 2020-07-31
埃克苏马生物技术公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These relatively long ex vivo expansion times create issues with cell viability and sterility, as well as sample consistency issues in addition to scalability challenges

Method used

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  • Methods and compositions for genetically modifying and expanding lymphocytes and regulating the activity thereof
  • Methods and compositions for genetically modifying and expanding lymphocytes and regulating the activity thereof
  • Methods and compositions for genetically modifying and expanding lymphocytes and regulating the activity thereof

Examples

Experimental program
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example

[0129] In certain aspects, provided herein are methods for performing adoptive cell therapy on an individual, as an illustrative example, the methods may include the following:

[0130] A. Collecting blood from an individual;

[0131] B. Isolation of peripheral blood mononuclear cells (PBMC) comprising resting T cells and / or resting NK cells;

[0132]C. Ex vivo contacting the individual's resting T cells and / or resting NK cells with a replication-defective recombinant retroviral particle comprising on its surface a protein capable of binding to resting T cells and / or NK cells and pseudotyped components that facilitate fusion of the replication-defective recombinant retroviral particle with its membrane, wherein the contacts facilitate transduction of quiescent cells with the replication-defective recombinant retroviral particle T cells and / or NK cells, thereby generating genetically modified T cells and / or NK cells; and

[0133] D. Reintroducing the genetically modified cell...

example 1

[1026] Example 1. Engineering of a retroviral packaging and transduction system to target resting T cells for selection of T cell integration and expression by PBMCs.

[1027] Although it is possible to generate high-titer lentiviral vectors using transient transfection, this method carries the risk of producing replication-deficient recombinant retroviruses (RCRs) and is not scalable for clinical applications. Here, a stable CAR gene was generated by synchronously introducing multiple constructs encoding inducible promoters and their regulators into HEK293 suspension-adapted cells (HEK293S) to stably produce viral components, CAR genes and their regulatory components. Retroviral packaging cell lines. Two different inducible systems can be used to temporarily control gene expression. One system is based on rapamycin- or rapamycin analog-induced dimerization of two transcription factors. One transcription factor consists of three copies of the FKPB protein fused to the ZFHD1 ...

example 2

[1031] Example 2. Production of lentiviral vectors and retroviral packaging.

[1032] With the construct for expressing Flp recombinase ( Figure 4C ) and the retroviral packaging stable cell line produced in Example 1 transfected with a construct containing a polynucleotide sequence encoding a CAR and under the control of the CD3Z promoter (which is inactive in HEK293S cells) The lymphoproliferative component IL7Rα-insPPCL, wherein CAR and IL7Rα-insPPCL are separated by a polynucleotide sequence encoding T2A ribosomal skipping sequence, and IL7Rα-insPPCL has ribonuclease controlled by acyclovir riboswitch. The CAR-containing construct further includes cPPT / CTS and RRE sequences and polynucleotide sequences encoding HIV-1 Psi. The entire polynucleotide sequence on the CAR-containing construct to be integrated into the genome is flanked by FRT sites. Successful integration of the CAR-containing construct resulted in constitutive expression of GFP that was thus removed by tran...

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Abstract

The present disclosure provides methods and compositions for genetically modifying lymphocytes and related methods that include genetically modifying T cells and / or NK cells. The methods use replication incompetent recombinant retroviral particles that comprise a pseudotyping element on their surface and optionally a membrane-bound T cell activation element, such as an anti-CD3, and encode one ormore engineered signaling polypeptides that can include a lymphoproliferative element, and / or a chimeric antigen receptor (CAR). The methods can include contacting PBMCs with replication incompetent recombinant retroviral particles for various exemplary time periods, such as less than 24 hours or in some illustrative embodiments less than 15 minutes. Illustrative chimeric lymphoproliferative elements that are capable of promoting survival and / or proliferation of T cells or NK cells in culture without adding IL-2, are provided. Furthermore, additional regulatory elements are provided, such as inhibitory RNA molecules.

Description

[0001] Cross References to Related Applications [0002] This application is a continuation-in-part of International Application No. PCT / US2018 / 020818, filed March 3, 2018; and claims the benefit of: U.S. Provisional Application No. 62 / 560,176, filed September 18, 2017; 2017 U.S. Provisional Application No. 62 / 564,253, filed September 27, 2017; U.S. Provisional Application No. 62 / 564,991, filed September 28, 2017; and U.S. Provisional Application No. 62 / 728,056, filed September 6, 2018 ; International Application No. PCT / US2018 / 020818 is a continuation-in-part of International Application No. PCT / US2017 / 023112 filed on March 19, 2017; International Application No. PCT / US2017 / 041277 filed on July 8, 2017 continuation-in-part of U.S. Application No. 15 / 462,855, filed March 19, 2017; and continuation-in-part of U.S. Application No. 15 / 644,778, filed July 8, 2017; and asserting Interest in: U.S. Provisional Application No. 62 / 467,039, filed March 3, 2017; U.S. Provisional Applicati...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C07K14/705C07K14/715A61K48/00C12N5/0783C07K14/725C12N15/867C12N5/10
CPCA61K48/00C12N15/86C12N2740/16043C12N2740/16045C12N2740/16052C12N2800/30C12N2830/205C12N2840/203C07K14/7051C07K14/70517C07K14/70532C07K14/71C07K14/7155C12N9/90C07K2319/02C07K2319/41C07K2319/43C07K2319/50C07K2319/60C07K2319/71C07K2319/81C12N5/0636C12N2510/00A61K39/4611A61K39/4631A61K39/464412A61K39/464402
Inventor 格雷戈里·伊恩·弗罗斯特詹姆斯·约瑟夫·奥努弗法扎德·哈里扎德
Owner 埃克苏马生物技术公司
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