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Chimeric antigen receptor

An antigen and ligand technology, applied in antibody medical components, fusion with soluble cell surface receptors, drug combinations, etc., can solve problems such as failure to cause long-term remission and loss of CD20 expression

Inactive Publication Date: 2019-08-09
AUTOLUS LIMIED
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, rituximab cannot be used against myeloma due to loss of CD20 expression in plasma cells
Newer agents such as bortezamib and lenolidomide are partially effective but fail to lead to long-term remission

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0414] Example 1 - Expression of BCMA on the surface of myeloma cells

[0415] Primary myeloma cells were isolated by CD138 immunomagnetic selection on fresh bone marrow samples from multiple myeloma patients known to have overt disease. These cells were stained with BCMA-specific J6MO mAb (GSK) conjugated to PE. In parallel, a standard of beads with a known number of binding sites was generated using the PE Quantibrite Bead Kit (Becton Dickenson) following the manufacturer's instructions. BCMA copy numbers on myeloma cells were obtained by correlating the mean fluorescence intensity from myeloma cells with a standard curve derived from the beads. The range of BCMA copy numbers on the surface of myeloma cells was found to be low: 348.7-4268.4 BCMA copies per cell, with a mean of 1181 and a median of 1084.9 ( figure 2 ). This is much lower than eg CD19 and GD2 (classic targets of CARs).

Embodiment 2

[0416] Example 2 - Design and Generation of TACI CAR

[0417] Immunization and hybridoma generation

[0418] The gene encoding human TACI was cloned in the vector pVAC2. Five BalBC mice were immunized with plasmid DNA encoding TACI adsorbed to gold nanoparticles. Using Gene-Gun TM (Biorad) system for intramuscular delivery of coated gold nanoparticles. Mice were boosted 3 times over the course of 21 days. Test blood from mice was screened for anti-TACI antibody titers by ELISA and flow cytometry.

[0419] Five mice with TACI-positive sera were selected for a final immune boost, and then spleens were harvested for B-cell isolation and hybridoma production. Hybridoma fusions using 10 x 96-well plates of lymphocytes from selected mice were performed. Hybridoma supernatants were screened for reactive anti-TACI antibodies by ELISA screening against recombinant human TACI purified protein (Peprotech, 310-17), representative data are shown in Figure 6a middle. ELISA positi...

Embodiment 3

[0427] Example 3 - Killing of target cells by TACI-CAR T cells

[0428] Retroviruses were generated by transiently transfecting 293T cells with plasmids encoding CAR, gag / pol, and the envelope protein RD114. After 3 days, the supernatant was harvested and used to transduce PHA / IL2-activated PBMCs at the same titer as the retrovirus on retronectin-coated plates. CAR expression was confirmed by flow cytometry 6 days after transduction, and PBMCs were co-cultured with TACI+BFP SupT1 cells or BCMA+BFP SupT1 cells at a ratio of 1:1. Target cell killing was determined after one and three days. Also after one and three days, supernatants were removed and interferon-γ levels were determined by ELISA. The result is as Figure 7 Shown in b and c.

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Abstract

The present invention provides a cell comprising first and second chimeric antigen receptors (CARs), which bind to different antigens, wherein the first CAR binds to Transmembrane activator and calcium modulator and cyclophilin ligand (CAML) interactor (TACI). The present invention also provides a cell comprising a tanCAR comprising first and second antigen-binding domains which bind to differentantigens, wherein the first antigen binding domain binds the antigen Transmembrane activator and calcium modulator and cyclophilin ligand (CAML) interactor (TACI). The present invention further provides corresponding nucleic acid sequences and / or constructs, kits and vectors comprising said nucleic acid sequences and / or constructs, molecules and methods for making such cells. The cells may be used in cellular immunotherapy approaches for treating diseases such as multiple myeloma.

Description

[0001] field of invention [0002] The present invention relates to cells comprising a chimeric antigen receptor (CAR) that binds an antigen transmembrane activator and a calcium modulator and cyclophilin ligand (CAML) interactor (TACI). Such cells can be used to treat cancerous diseases such as multiple myeloma. [0003] Background of the invention [0004] Chimeric Antigen Receptor (CAR) [0005] Chimeric antigen receptors are proteins that, in their usual form, graft the specificity of monoclonal antibodies (mAbs) to the effector functions of T cells. Their usual form is a type I transmembrane domain protein with an amino-terminus-recognizing antigen, a spacer, a transmembrane domain, all linked to complex intracellular domains that transmit T-cell survival and activation signals (see figure 1 ). [0006] The most common forms of these molecules use single-chain variable fragments (scFv) derived from monoclonal antibodies to recognize target antigens. The scFv is fused t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28
CPCC07K16/2878A61K2039/5156A61K2039/55A61K2039/585C07K2317/31C07K2317/622C07K2319/03C07K2319/33A61P35/00C07K2319/32
Inventor S.奥努奥哈V.佩达雷迪加里L.斯坦克祖克M.费拉里B.马
Owner AUTOLUS LIMIED
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