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Chimeric antigen receptors and gene editing of cd2 for immunotherapy of t-cell malignancies

A chimeric antigen receptor, cell technology for targeting specific cell fusion, receptor/cell surface antigen/cell surface determinant, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, etc. direction

Inactive Publication Date: 2021-01-01
NAT UNIV OF SINGAPORE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Reports using TALEN deletion of the TCRα and CD52 loci together with anti-CD19 CAR expression suggest that combining CAR expression with gene editing is feasible in the clinical setting, but is technically challenging

Method used

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  • Chimeric antigen receptors and gene editing of cd2 for immunotherapy of t-cell malignancies
  • Chimeric antigen receptors and gene editing of cd2 for immunotherapy of t-cell malignancies
  • Chimeric antigen receptors and gene editing of cd2 for immunotherapy of t-cell malignancies

Examples

Experimental program
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example 1

[0275] Example 1: Blocking CD2 Expression in T Cells for Effective Chimeric Antigen Receptor Therapy

[0276] This embodiment illustrates the use of a novel method combined with a second-generation CAR to block CD2 expression, thereby generating effective anti-CD2 CAR-T cells. This practical strategy provides a new treatment option for cancer patients.

[0277] figure 1 Exemplary anti-CD2 chimeric antigen receptors (CARs) are provided. The scFv of anti-CD2 monoclonal antibody 9.6 was linked to the CD8α signal peptide, the CD8α hinge transmembrane domain, and the intracellular domains of anti-CD19-4-1BB-CD3 CAR4-1BB and CD3ζ previously developed in the laboratory. The scFv of anti-CD2 monoclonal antibody 9.6 or 9-1 was linked to the CD8α signal peptide, and the sequence encoding the localization domain and optionally the CD8α hinge transmembrane domain. They were subcloned into murine stem cell virus (MSCV) vectors. In certain instances, the MSCV is an MSCV internal ribosom...

example 2

[0285] Example 2: Genetic modification of CD2 in leukemia cells

[0286] This example illustrates the downregulation of endogenous CD2 expression in leukemia cells using a CRISPR-based platform.

[0287] These gRNA sequences were used with WT SpCas9 to introduce DSBs for genome editing. These sgRNA sequences are described and validated in Sanjana et al., Nat Methods, 2014 Aug, 11(8):783-4.

[0288] Table 2. Exemplary guide RNA target sequences.

[0289] SEQ ID NO: gRNA name gRNA target sequence SEQ ID NO: 44 CD2 CRISPR guide RNA 1 CAAAGAGATTACGAATGCCT SEQ ID NO: 45 CD2 CRISPR guide RNA 2 CTTGTAGATATCCTGATCAT SEQ ID NO: 46 CD2 CRISPR guide RNA 4 CACGCACCTGGACAGCTGAC SEQ ID NO: 47 CD2 CRISPR guide RNA 5 GGTTGTGTTGATACAAGTCC

[0290] lentiviral construct

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Abstract

The present invention provides gene edited or gene silenced immune cells comprising an chimeric antigen receptor (CAR) such as an anti-CD2 CAR. In some embodiments, such engineered immune cells lack CD2 expression. Also, provided herein are methods of using such cells in cancer therapies.

Description

[0001] Cross References to Related Applications [0002] This application claims priority to U.S. Provisional Application No. 62 / 675,525, filed May 23, 2018, the contents of which are incorporated herein by reference in their entirety. [0003] References to Sequence Listings [0004] This application contains a Sequence Listing, which is filed electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy, created on May 23, 2019, is named "119419_5005_WO_SequenceListing_ST25.txt" and is 44.0KB in size. [0005] field of invention [0006] The invention described herein generally relates to clinically effective populations of chimeric antigen receptor T cells (CAR-T cells) lacking CD2 expression through CD2 gene modification. The present invention also relates to the use of such CAR-T cells in the treatment of T cell malignant tumors. Background technique [0007] Genetically engineered immune cells are powerful new treatments for c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783C07K16/28A61K39/00A61P35/02
CPCA61P35/00A61K47/65A61K47/66C07K16/2806C12N2310/20C12N15/86C07K2317/622A61K39/4611A61K39/464466A61K39/4631C12N5/0638A61P35/02C07K2319/03C07K2319/33C07K14/7051C12N2510/00C07K14/70507C12N5/0636C07K14/70517C07K14/70578C07K2319/02
Inventor 达里奥·坎帕纳
Owner NAT UNIV OF SINGAPORE