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Methods for selectively modulating the activity of distinct subtypes of cells

A target cell, cell targeting technology, applied in the active field of different subtypes, can solve the problem of expensive culture system, achieve the effect of improving efficiency and preventing in vivo resistance problems

Pending Publication Date: 2019-04-16
ECOLE NORMALE SUPERIEURE DE LYON +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This culture system is very expensive as it requires regular cytokine supplementation (every two days)

Method used

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  • Methods for selectively modulating the activity of distinct subtypes of cells
  • Methods for selectively modulating the activity of distinct subtypes of cells
  • Methods for selectively modulating the activity of distinct subtypes of cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0432] Example 1: Selective activation of CD4+ T cells

[0433] To activate CD4 + Lymphocytes do not activate CD8 + Lymphocytes, generated VLPs that presented CD4-specific DARPins as targeting ligands and IL-7 as activation domains on the MV H protein as well as the fusion protein (F), resulting in 4 H / IL7 H - VLPs (proteins comprising the sequences SEQ ID NO: 2, 14 and 15). To generate particles, a transfection protocol was established. Briefly, 0.45 μg of pCG-Hmut-CD4-DARPin (Zhou et al., J Immunol, 2015), 0.45 μg of pCG-HΔ15-IL7 (provided by Els Verhoeyen), 4.7 μg of pCG-FcΔ30 (Funke et al., Molecular therapy, 2008), 14.4 μg of HIV-1 packaging plasmid pCMVΔR8.9 (Funke et al., Molecular therapy, 2008) and 15.1 μg of pCG-1 were transfected into HEK293T cells in a T175 flask. After harvesting, the carrier particles are further concentrated by ultracentrifugation. A titer of ~10 LV was obtained 7 tu / ml, a VLP titer of ~1.5 μg p24 / ml was obtained.

[0434] 4 was confi...

Embodiment 2

[0436] Example 2: Delivery of tumor-specific chimeric antigen receptors into resting T cells

[0437] Since IL7-presenting vectors trigger activation of resting T cells, these cells are expected to allow lentiviral transduction. To demonstrate this, two versions of the 4 H / IL7 H -LV delivery of the GFP transgene. Such as Figure 9 shown, 4 H / IL7 HΔ15 -LV and 4 H / IL7 HΔ20 -LVs can efficiently and selectively transduce resting CD4 in cell mixtures + T cells.

[0438] Chimeric antigen receptors (CARs) are powerful tools for cancer therapy. Until now, for the genetic delivery of CARs, T cell subsets had to be purified and activated. Here, it is demonstrated that CAR can be selectively delivered to resting CD4 + in cells. The particles were generated as described in Example 1, except that pCG-1 was replaced by a CAR-encoding transfer plasmid. When resting T cells are delivered with 4 H / IL7 H - When transduced with ErbB2-specific chimeric antigen receptor (ErbB2...

Embodiment 3

[0439] Example 3: MV glycoproteins can be replaced by NiV's MV glycoproteins to selectively activate and transduce resting human CD8 + T cells

[0440] To efficiently pseudotype VLPs or LVs with NiV glycoproteins, the cytoplasmic tail of NiV-G was truncated by 34 amino acids (GcΔ34) and that of NiV-F by 22 amino acids (FΔ22). Next, the natural receptor recognition of Ephrin-B2 / B3 by GcΔ34 was disrupted by introducing four point mutations (E501A, W504A, Q530A, E533A) into GcΔ34. LVs pseudotyped with this engineered G protein completely lost binding to native Ephrin-B2 and Ephrin-B3NiV receptors ( figure 2 ), and do not enter cells expressing these receptors.

[0441] Furthermore, NiV-pseudotyped LVs have some attractive features, such as high yield and resistance to intravenous immunoglobulin. Since there is no vaccination against NiV and the few outbreaks are limited to a few cases in Malaysia, Bangladesh and India (SEARO|WHO Southeast Asia Region), neutralizing antibodi...

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Abstract

The present invention relates to pseudotyped retrovirus-like particles or retroviral vectors comprising both engineered envelope glycoproteins derived from a virus of the Paramyxoviridae family fusedto a cell targeting domain and fused to a functional domain. The present invention also relates to the use of said pseudotyped retrovirus-like particles or retroviral vectors to selectively modulate the activity of specific subsets of cells, in particular of specific immune cells. These pseudotyped retrovirus-like particles or retroviral vectors are particularly useful for gene therapy, immune therapy and / or vaccination.

Description

technical field [0001] The present invention relates to methods for selectively modulating the activity of different subtypes of cells. Background technique [0002] Many types of pathology involve the cells of the immune system. Therefore, enhancing or reducing their activity is the focus of many therapeutic strategies. Various types of immune cells with very different functions can be distinguished by the expression of certain cell surface proteins. To date there is no technology capable of selectively activating or inactivating only individual cell subtypes, especially in vivo. [0003] The use of cytokines to induce or promote the generation of a desired immune response is an attractive approach in immunotherapy against cancer. Cytokines are often administered as non-specific auxiliary substances in support of other immunotherapies or as a supplement to chemotherapy. But so far, cytokine-based therapy has rarely been used due to unresolved systemic toxicity. Further...

Claims

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Application Information

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IPC IPC(8): C12N15/86
CPCC12N7/00C12N15/86C07K14/005A61K39/12A61P21/00A61P25/00A61P29/00A61P3/00A61P31/00A61P35/00A61P35/02A61P37/02A61P37/08A61P43/00C12N2740/16023C12N2740/16043C12N2760/18422C12N2760/18222C12N2740/16045C07K2319/33C07K2319/74C07K2319/30C07K2319/03C12N2760/18434C07K14/5418C07K16/2812C07K16/2815C07K16/32A61K2039/505C07K2317/622A61K2239/57A61K39/464412A61K39/4631A61K39/4644A61K39/464838A61K2239/31A61K39/464444A61K39/464406A61K39/4611C07K14/7051
Inventor 卡洛琳·科斯塔·费霍兹埃尔斯·维霍伊恩弗朗索瓦-卢瓦克·科塞鲁本·本德尔克里斯蒂安·巴克霍尔兹周琦
Owner ECOLE NORMALE SUPERIEURE DE LYON
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