Chimeric antigen receptor

一种嵌合抗原受体、抗原的技术，应用在抗体模拟物/支架、杂合肽、药物组合等方向，能够解决不可预测、冗长、不成功等问题

Active Publication Date: 2017-11-28
AUTOLUS LIMIED
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This is a lengthy and often unsuccessful approach that can lead to unpredictable results and increases the risk of off target binding

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0375] Example 1 - COMP CAR Expression on the Cell Surface

[0376] Anti-CD33COMP CAR (amino acid sequence shown in Figure 5 c and the nucleic acid sequence are shown in Figure 5 d) or anti-CD33 IgG1 CAR transduced murine T cell lines.

[0377] These cells were then stained with chimeric soluble CD33 fused to murine Fc IgG2a, followed by secondary staining with anti-mouse IgG PE ( Figure 6 a).

[0378] Use anti-ROR-1COMP CAR CAR (amino acid sequence shown in Figure 5 c and the nucleic acid sequence are shown in Figure 5 d) or anti-ROR-1 IgG1 CAR transduced murine T cell lines.

[0379] These cells were then stained with soluble His-tagged ROR-1, followed by secondary staining with anti-His-biotin, and then triple staining with streptavidin-TS3 ( Figure 6 b).

[0380] All four CARs were successfully expressed on the cell surface. These data also suggest that when linked to the COMP spacer, the CAR-binding domain is oriented in a manner that does not impede ligan...

Embodiment 2

[0381] Example 2 - Stimulation of COMP CAR T cells using immobilized ligands

[0382] COMP ROR-1 CAR T cells were stimulated with T cells and with immobilized ligand-coated beads. To achieve this, a soluble His-tagged ROR-1 was constructed and expressed. The supernatants containing these soluble ligands were then incubated with a certain number of anti-His beads at different concentrations. The beads were then washed to remove unbound ligand, and these beads were used to stimulate T cells transduced with the COMPCAR platform or an equivalent CAR with an IgG spacer.

[0383] Transduced murine T cells were co-cultured with anti-His beads pre-coated with different concentrations of soluble His-tagged ROR-1 supernatant. After 16-24 hours, the amount of IL-2 in the co-culture supernatant was analyzed by ELISA ( Figure 7 ).

Embodiment 3

[0384] Expression level of embodiment 3-ROR-1 target cells

[0385] The SKW cell line naturally expresses low levels of ROR-1. These cells were transduced with ROR-1 to increase expression levels. These cells were stained with anti-ROR-1APC and compared with unstained cells ( Figure 8 ).

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Abstract

The present invention provides a chimeric antigen-receptor (CAR)-forming polypeptide comprising: (i) an antigen-binding domain; (ii) a coiled-coil spacer domain; (iii) a transmembrane domain; and (iv) an endodomain. The invention also provides a multimeric CAR formed by association of a plurality of CAR- forming polypeptides by virtue of association of their coiled-coil spacer domains.

Description

field of invention [0001] The present invention relates to chimeric antigen receptors (CARs) comprising specific spacer domains leading to the formation of multimeric CAR molecules on the cell surface. The multimeric CAR molecule may be "ultrasensitive" and capable of inducing T cell activation in response to bound antigen expressed at low density on target cells. Background of the invention [0002] Chimeric Antigen Receptor (CAR) [0003] Antigen-specific T cells have traditionally been generated by the selective expansion of peripheral blood T cells naturally specific for the target antigen. However, it is difficult and often impossible to select and expand large numbers of T cells specific for most cancer antigens. Gene therapy using integrating vectors offers a solution to this problem, since transgenic expression of chimeric antigen receptors (CARs) allows generation of antibodies against any surface by ex vivo viral vector transduction of bulk populations of periphe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00A61K35/17A61P35/00
CPCC07K14/705C07K2319/00C07K2319/03A61K39/4611A61K39/4631A61K39/464412A61K2239/24A61K39/464462C12N5/0636A61K39/464402C12N5/0646A61P35/00A61P35/02A61P37/04A61P43/00C07K16/28C07K2317/622C12N2510/00A61K35/17
Inventor M.普莱S.科尔多巴
Owner AUTOLUS LIMIED
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