IP Library Granted Patent US 8,999,715
Granted Patent B2
US 8,999,715 · App. 13/240,009 · Granted Apr 7, 2015

Use of common γ chain cytokines for the visualization, isolation and genetic modification of memory T lymphocytes

Inventors: Maria Chiara Bonini (Peschiera Borromeo, IT); Attilio Bondanza (Melzo, IT)
Assignee: Ospedale San Raffaele S.R.L.
G01N33/56972A61K48/0091C12N5/0636G01N33/6863A61K2039/5156C12N2501/23
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Quick Facts
Patent No.
US 8,999,715
App. No.
13/240,009
Granted
Apr 7, 2015
Kind
B2
Abstract

It is described in vitro methods for expanding, detecting or isolating rare populations of antigen specific memory T cells. It is also described an in vitro method for obtaining a genetically modified memory T cell population. Uses of cells so obtained are also disclosed.

Claims (14)

1. An in vitro method for obtaining a genetically modified memory T cell population that is CD4+ or CD8+, comprising the steps of:

a) activating lymphocytes in vitro in a cell-free medium with at least two specific activating receptor agonist antibodies that are able to drive lymphocyte activation, wherein one of the lymphocyte activating receptor agonist antibodies is specific for CD3 polypeptide and the other lymphocyte activating receptor agonist antibody is specific for CD28;

b) exposing activated lymphocytes in vitro, in a cell-free medium, to an effective amount of interleukin added to the medium, wherein the interleukin is at least IL-7 and IL-15, able to selectively expand populations of memory T cells; and

c) inserting and expressing an exogenous gene by means of an appropriate vector into the lymphocytes as obtained in b) to produce a genetically modified memory T cell population that is CD4+ or CD8+;

wherein the effective amount of the interleukin added to the medium is from 5 ng/ml to 50 ng/ml;

wherein the medium and interleukin of step b) are replaced every 3 to 4 days; and

wherein about 80% of both CD8+ and CD4+T− cells of steps b) and c) are positive for the marker CD62L and marker CD127 and are central memory T-lymphocytes.

2. The in vitro method of claim 1 wherein said lymphocytes are derived from a biological sample selected from the group consisting of: blood and other liquid samples of biological origin, solid tissue samples, tissue cultures of cells derived therefrom and the progeny thereof, isolated cells from biological samples.

3. The in vitro method of claim 1 wherein the specific lymphocyte activating receptor agonist is conjugated to cell-mimicking cell-free supports.

4. The in vitro method of claim 3 wherein the cell-mimicking supports are paramagnetic beads.

5. The in vitro method of claim 1 wherein the vector is a viral vector.

6. The in vitro method of claim 5 wherein the exogenous gene comprises a gene selected from the group consisting of a suicide gene, a marker gene, a biologically active molecule, a receptor, a soluble factor retained in the cell or released outside the cell, a gene conferring resistance to a prodrug and combinations thereof.

7. The method of claim 1 wherein the effective amount of the interleukin added to the medium is 5 ng/ml, or 50 ng/ml.

8. The method of claim 1 wherein the interleukin added to the medium is exogenous interleukin.

Assignments (2)
TRANSFER OF BUSINESS Recorded Feb 5, 2013
From: FONDAZIONE CENTRO SAN RAFFAELE DEL MONTE TABOR
To: OSPEDALE SAN RAFFAELE S.R.L.
Reel/Frame 029752/0277 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Dec 7, 2011
From: BONINI, MARIA CHIARA; BONDANZA, ATTILIO
To: FONDAZIONE CENTRO SAN RAFFAELE DEL MONTE TABOR
Reel/Frame 027339/0500 →
Continuity (3)
Continuation 12063373
Provisional Application 60706503 · Aug 8, 2005
Related Publication 20120027802A1 · Feb 2, 2012