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Ror1(ntrkr1)specific chimeric antigen receptors for cancer immunotherapy

A chimeric antigen receptor and specific technology, applied in the direction of targeting specific cell fusion, medical raw materials derived from mammals, antibodies, etc., can solve the problem of increasing the complexity of initial risk stratification

Active Publication Date: 2017-07-04
CELLECTIS SA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Thus, the complexity of initial risk stratification for CLL and treatment has increased significantly
Furthermore, the approach to CLL patients with fludarabine-refractory disease can be quite challenging when these initial treatments are ineffective (Byrd J.C et al., 2014)

Method used

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  • Ror1(ntrkr1)specific chimeric antigen receptors for cancer immunotherapy
  • Ror1(ntrkr1)specific chimeric antigen receptors for cancer immunotherapy
  • Ror1(ntrkr1)specific chimeric antigen receptors for cancer immunotherapy

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0371] Example 1: Screening of ROR1 positive and negative cell lines

[0372] To identify cell lines expressing different cell surface expression levels of ROR1, 12 human cell lines were analyzed by flow cytometry using Qifikit (Dako) and anti-human ROR1 mAb clone 2A2 (see Materials and Methods). Nine of these cell lines have been previously described in the literature to be positive for ROR1 at the mRNA or protein level (Table 10).

[0373] Table 10: Cell lines positive for ROR1 reported in the literature

[0374] cell line describe cell type MDA-MB-231 Adhesive breast cancer PC-3 Adhesive Prostate adenocarcinoma MDA-MB-468 Adhesive breast cancer Hs 746T Adhesive stomach cancer NCI-H1993 Adhesive non-small cell lung cancer HT-29 Adhesive colorectal adenocarcinoma A549 Adhesive human lung adenocarcinoma Cal51 Adhesive breast cancer Jeko-1 floating mantle cell lymphoma

[0375] Fl...

Embodiment 2

[0379] Example 2: Production of anti-ROR1 scCAR

[0380] A panel of 18 second-generation ROR1-specific scCARs was generated and tested in the following experiments ( figure 1 C).

[0381] They are produced by the fusion of the building blocks of Table 1-2 below:

[0382] - a scFv from murine origin or a humanized form from D10, G6, G3, H10, 2A4 or 1C11;

[0383] - a spacer: human FcγRIIIα, CD8α or IgG1 hinge;

[0384] - the transmembrane domain of human CD8α and the co-stimulatory domain of human 41BB;

[0385] - Activation domain of human CD3ζ

[0386] Different scFvs are used in scCARs to generate receptors with different binding affinities and different epitope specificities.

[0387] mAbs D10 and G6 target the 3' Ig-like region and the linker region between the Ig-like domain and the CRD domain of ROR1 ( figure 2 )

[0388] mAbs G3, H10 and 2A4 target the IgG-like region of ROR1 ( figure 2 )

[0389] mAb 1C11 targets the CRD domain of ROR1 ( figure 2 ).

[0390...

Embodiment 3

[0391] Example 3: In vitro testing of anti-ROR1 scCAR

[0392] In human primary T cells, use the Figure 6 The two-step screening presented in tested previously designed anti-ROR1 scCARs.

[0393] In the first step of the screening method described, primary human T cells preactivated for 4-5 days with anti-CD28 / CD3 beads and IL-2 were treated with mRNAs encoding the 18 anti-ROR1 scCARs presented earlier. Cell electroporation. One day after electroporation, scCAR expression was assessed by flow cytometry and western blotting, and scCAR-modified T cell activity was assessed by measuring T cell degranulation. ScCARs detected by Western blot and that induced significant specific T cell degranulation (≥20%) when co-cultured with at least one ROR1-positive cell line were selected to undergo the second step of the screening method.

[0394] In the second step of the screening method, primary human T cells previously activated for 11-12 days with anti-CD28 / CD3 beads and IL-2 were t...

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Abstract

The present invention relates to Chimeric Antigen Receptors (CAR) that are recombinant chimeric proteins able to redirect immune cell specificity and reactivity toward selected membrane antigens, and more particularly in which extracellular ligand binding is a scFV derived from a ROR1 monoclonal antibody, conferring specific immunity against ROR1 positive cells. The engineered immune cells endowed with such CARs are particularly suited for treating lymphomas and leukemia, and for solid tumors such as breast, colon, lung, and kidney tumors

Description

[0001] field of invention [0002] The present invention relates to chimeric antigen receptors (CARs), which are recombinant chimeric proteins capable of redirecting immune cell specificity and reactivity to ROR1, which is found on most myeloid cells and used for diagnosis in patients Cell surface glycoproteins of chronic lymphocytic leukemia (CLL) or solid tumors such as breast, colon, lung and kidney tumors. When it is expressed in T cells or NK cells, the CAR according to the present invention is particularly useful for the treatment of malignant cells bearing the ROR1 antigen. The resulting engineered immune cells display high levels of specificity for malignant cells, conferring safety and efficacy to immunotherapy. [0003] Background of the invention [0004] Adoptive immunotherapy, which involves the transfer of ex vivo generated autologous antigen-specific T cells, is a promising strategy for the treatment of viral infections and cancer. T cells for adoptive immunoth...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N5/10A61K35/17A61P37/00
CPCC07K14/7051C07K14/70517C07K16/2803C07K16/30C07K2319/03C07K2319/02C07K2317/622A61K39/4631C12N5/0636A61K39/464462A61K39/4611A61K2039/505A61K2039/572C12N2740/15043C07K2317/24C07K2317/53C07K2319/33C12N2510/00C12N15/62A61P35/00A61P37/00A61P37/04A61K48/00A61K2039/5158A61K2039/5156A61K35/17A61K39/00C07K19/00C12N5/10
Inventor C·西弗-曼尼奥伊
Owner CELLECTIS SA
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