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Methods and compositions for promoting immune cell function

A composition, technology of nucleated cells, applied to the preparation of a frozen composition of nucleated cells, the preparation and/or thawing of a frozen composition of nucleated cells, the preservation of the cellular function of nucleated cells, the preparation of a thawed composition of nucleated cells field

Inactive Publication Date: 2019-05-03
TORQUE THERAPEUTICS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Limitations of existing immune cell isolation, storage, freezing, thawing and / or expansion protocols remain

Method used

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  • Methods and compositions for promoting immune cell function
  • Methods and compositions for promoting immune cell function
  • Methods and compositions for promoting immune cell function

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0661] Example 1: Formation of protein nanogels

[0662] Proteins were produced from suspension-adapted HEK 293 cells in serum-free medium, followed by affinity purification by protein A and buffer exchange into Darby's phosphate-buffered saline (DPBS).

[0663] Backpack protein nanogels comprising cross-linked protein nanogels (backpacks) were formed as follows. The proteins or protein complexes described herein are crosslinked into protein nanoparticles using an excess of degradable crosslinkers. A suitable protein complex is the IL-15 superagonist described in Rubinstein et al., PNAS 103:24p.9166-9171 (2006).

[0664] In some embodiments, the protein complex comprises the wild-type human IL-15 protein sequence as follows:

[0665]

[0666] A suitable crosslinker is bis[2-(N-succinimidyl-oxycarbonyloxy)ethyl]disulfide, which contains two N- Hydroxysuccinimide (NHS) ester group.

[0667] After incubation at room temperature, the reactions were diluted with DPBS to the ...

Embodiment 2

[0669] Example 2: Isolation of T cells, B cells and NK cells

[0670]Isolation of T cells and NK cells from healthy donors. One-day-old leukopack cells (Biospecialties, Inc.) were diluted 1:1 in volume with DPBS and plated on density pads (Lymphoprep, Stemcell Tech.) in 50 ml Tubes (35 ml diluted leukopack on top of 15 ml lymphocyte separation reagent) were layered. After centrifugation at 800g for 30 minutes, monocytes were harvested at the interface between the lymphocyte separator and DPBS. Cells were washed 3 times in 50 ml DPBS to remove residual lymphocyte separation agent and cell debris. T cells and NK cells were isolated by serial magnetic bead sorting using anti-CD3 (or anti-CD8) and anti-CD56 conjugated beads (Miltenyi), respectively, according to the manufacturer's instructions. Briefly, LS columns were equilibrated with 3 ml ice-cold DPBS, while antibody-conjugated beads were incubated with monocytes (30 min at +4°C). After loading the cells into the column, w...

Embodiment 3

[0672] Example 3: Activation of T cells prior to labeling

[0673] Before binding to protein nanogels, pooled CD4 + and CD8 + T cells (which are the major cell type produced by the anti-CD3 selection described in Example 2) or isolated CD8 + T cells (both obtained as described in Example 2) were first activated with CD3 / CD28 Dynabeads (ThermoFisher, Cat# 1132D) according to the manufacturer's instructions. Incubate T cells and CD3 / CD28 Dynabeads in CM-T supplemented with 20 ng / ml interleukin-2 (IL-2) at 37°C and 5% CO 2 Incubate for 2 days. CD3 / CD28 Dynabeads were then removed from T cells by magnetic separation.

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Abstract

The present disclosure features, at least in part, methods for conserving cell function, immune cell function, e.g., after one or more cycles of freezing and / or thawing the nucleated cell. In embodiments, the methods comprise contacting an immune cell with a protein nanoparticle comprising an IL-15 complex.

Description

[0001] related application [0002] This application claims priority to US Application Serial No. 62 / 349,473, filed June 13, 2016, which is incorporated herein by reference in its entirety. Background technique [0003] Immune cell therapy, such as adoptive cell therapy (ACT), involves the steps of collecting immune cells from a subject, expanding the cells, and reintroducing the cells into the same subject or a different subject. For example, ACT of donor-derived, ex vivo-expanded human cytotoxic T lymphocytes (CTLs) has emerged as a promising cancer therapy. Examples of ACTs include cultured tumor infiltrating lymphocytes (TILs), isolated and expanded T cell clones, and genetically engineered lymphocytes (eg, T cells) expressing conventional T cell receptors or chimeric antigen receptors. Genetically engineered lymphocytes designed to eliminate cancer cells expressing specific antigens are expanded and delivered to patients. ACT can provide tumor-specific lymphocytes (eg, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/20A61K47/54A61K47/68A61K38/17
CPCA61K38/00A61K38/1793A61K38/2086C07K2319/30A61K47/68A61K47/6901A61P11/06A61P35/00A61P3/10A61P37/06A61P37/08A61P9/00C07K14/54C07K14/56C07K14/565C07K14/70514C07K14/70517C07K14/70553C07K14/70589C07K2317/52
Inventor F·普奇T·L·安德烈森D·S·琼斯U·尼尔森J·A·拉克斯特罗
Owner TORQUE THERAPEUTICS INC
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