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Reducing fratricide of immune cells expressing nkg2d-based receptors

A technology of immune cells and receptors, applied in the field of immunotherapy, can solve problems such as poor cell yield

Pending Publication Date: 2021-01-12
CELYAD SA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Indeed, it has been observed that both scale-up and cryopreservation of T cells expressing chimeric NKG2D receptors lead to poor cell yields, presumably due to the effects of suicide or killing agents

Method used

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  • Reducing fratricide of immune cells expressing nkg2d-based receptors
  • Reducing fratricide of immune cells expressing nkg2d-based receptors
  • Reducing fratricide of immune cells expressing nkg2d-based receptors

Examples

Experimental program
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Effect test

Embodiment 1

[0186] Example 1. NKR-2 CAR T cells undergo killing of killers, which drive the phenotype of engineered T cell populations and amplification.

[0187] Following transduction and in vitro culture, NKR-2 T cell populations display predominant CD8 compared to T cells transduced with control vectors in the absence of means to control for killer + T cell subset composition (truncated CD19 (tCD19), figure 1 A). NKG2D expression was not restricted to NKR-2 T cells but was also clearly seen on control tCD19 T cells, although engagement of endogenous NKG2D failed to generate therapeutic responses in CAR T cells (32). However, the relative cell surface expression of NKG2D was highly increased in the NKR-2 T cell population, suggesting that T cells were transduced with the CAR construct (Figure 1B). The mean fluorescence intensity of NKG2D(CD314) was in CD4 + and CD8 + NKR-2 in both subpopulations was significantly higher in T cell populations, confirming CAR expression in both s...

Embodiment 2

[0194] Example 2. PI3K inhibition improves NKR-2 T cell viability and drives NKR-2 antigen during cryopreservation Increased production of specific cytokines and increased memory phenotype.

[0195] Following ligand binding, NKG2D and its associated DAP10 initiate signaling through the PI3K pathway in a manner similar to CD28 (25, 26). We therefore questioned whether inhibition of PI3K signaling could abrogate NKR-2-mediated killing during T cell culture. To this end, increasing concentrations of LY294002 (as a broad PI3K inhibitor) were added to the transduction and amplification phases of NKR-2 production.

[0196]The addition of LY294002 led to several observations. First, the cell surface expression of NKR-2T on NKR-2 T cells decreased in a dose-dependent manner, reaching the level of control tCD19 T cells at 10 μM ( Figure 4 A and Figure 5 ). This reduction was reversible, as removal of LY from cultures resulted in increased NKG2D expression up to the level of u...

Embodiment 3

[0199] Example 3. Antibody-mediated NKG2D blockade prevents NKR-2 CAR T cell killing.

[0200] Initial experiments including an anti-NKG2D antibody (clone 1D11) during NKR-2 T cell culture showed that NKR-2 T cell yields at the end of culture were comparable to control T cells (2.6-fold expansion of NKR-2 T cells, NKR- 2 T cells have a 13.8-fold expansion of antibody blocking ability ( Figure 8 ). This suggests that antibody blockade can abolish NKG2D target-driven killers. Dose titration experiments showed that antibody concentrations of 2.5 μg / mL and above could protect tCD19 T cells358 from NKR-2 T cell-targeted killing ( Figure 4 E). Anti-NKG2D antibody can also effectively block the release of IFN-γ in the process of target cell participation ( Figure 4 F), thereby confirming the specificity of NKR-2 T cells. Efficient killing of killers using anti-NKG2D antibodies is further supported by the fact that the observed IFN-γ release during NKR-2 T cell generation, l...

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Abstract

The present application relates to the field of immunotherapy, more particularly to the manufacture of cells for adoptive cell therapy. Provided herein are methods to prevent and / or reduce fratricideduring manufacturing of such cells, particularly of cells expressing a chimeric NKG2D receptor. Also provided are cells and compositions comprising cells in which fratricide is prevented and / or reduced.

Description

technical field [0001] The present invention relates to the field of immunotherapy, and more particularly to the manufacture of cells for adoptive cell therapy. Provided herein are methods of preventing and / or reducing killer agents during the production of such cells, particularly cells expressing NKG2D receptor cells. Cells and compositions comprising cells that prevent and / or reduce killers are also provided. Background technique [0002] Improvements in our understanding of the immune system have led to the development of many immune-targeted therapies that are currently delivering objective clinical responses in patients with advanced cancer. One of these approaches is chimeric antigen receptor (CAR) T cells, in which a patient's T cells are genetically modified to express a tumor-targeted CAR and then returned to the patient in large numbers (1). This type of adoptive cell therapy has reached a level of validation through objective clinical responses in patients with...

Claims

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Application Information

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IPC IPC(8): C12N5/0783C07K14/725C07K14/705C07K14/47A61K35/17
CPCC12N5/0636C12N2501/727C12N2501/998C12N2510/00C07K14/7056C12N15/1138C12N2310/14C12N2310/20C12N2310/531C12N2310/3233A61K38/177A61K39/4611A61K39/4631A61K39/464429A61P35/00A61K35/17
Inventor D·吉勒姆E·布雷曼B·德莫林S·博恩沙因S·拉伊塔诺
Owner CELYAD SA
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