A method to up-regulate cancer stem cell markers for the generation of antigen specific cytotoxic effector t cells

Patent metadata

Field	Value
Publication number	US-2017191034-A1
Application number	US-201515508897-A
Country	US
Kind code	A1
Filing date	Sep 4, 2015
Priority date	Sep 4, 2014
Publication date	Jul 6, 2017
Grant date	—

Abstract

The invention concerns a method of preparing a composition comprising stimulated immune system cells such as dendritic cells (DC) for use in inducing immune response of cytotoxic T lymphocytes against colorectal cancer. The dendritic cells are pulsed by contact with colorectal cancer stem cells (CSC) or their fragments thereof. These colorectal CSCs are produced by OSKM (Oct4, Sox2, Klf4 and c-Myc) induced reprogramming of differentiated colorectal cancer cells (CRC) which results in undifferentiated colorectal CSC-like cells. Both the CSC-like cells and the lysates of heat-shocked CSC-like cells could be used as an accessible source of tumour antigens for DC pulsing to induce specific immune responses against colorectal CSCs.

First claim

1 .- 41 . (canceled)

42 . A composition comprising at least one isolated dendritic cell loaded with at least one colorectal cancer stem cell, or fragments thereof, wherein the colorectal cancer stem cell comprises a differentiated colorectal tumour cell that has been reprogrammed with a baculoviral vector into an undifferentiated colorectal stem-cell state, wherein the baculoviral vector comprises zinc-finger nuclease-coding sequences and a fusion gene comprising a cell reprogramming factor or transcription factor capable of reverting or reprogramming the differentiated colorectal tumour cell into the undifferentiated colorectal cancer stem-cell state.

43 . The composition of claim 42 , wherein the dendritic cell is a mature dendritic cell.

44 . The composition of claim 43 , wherein the mature dendritic cell comprises an immature dendritic cell that has been loaded with said at least one colorectal cancer stem cell, or fragments thereof.

45 . The composition of claim 44 , wherein the mature dendritic cell has upregulated CD83, CD40 and CD86 expression.

46 . The composition of claim 42 , wherein the colorectal cancer stem cell is an in vitro (test-tube) colorectal stem cell that has been reprogrammed in vitro.

47 . The composition of claim 42 , wherein the colorectal cancer stem cell is a heat-shocked colorectal cancer stem cell, or fragments thereof.

48 . The composition of claim 47 , wherein the fragment is a lysate of a heat-shocked colorectal cancer stem cell.

49 . The composition of claim 42 , wherein the cell reprogramming factor or transcription factor is selected from the group consisting of Oct4, Sox2, Klf4 and c-myc.

50 . A composition for stimulating the immune system of a subject comprising at least one dendritic cell loaded with one or more antigens of a colorectal cancer stem cell, wherein said colorectal cancer stem cell comprises a differentiated colorectal tumour cell that has been reprogrammed with a baculoviral vector into an undifferentiated colorectal stem-cell state, wherein the baculoviral vector comprises zinc-finger nuclease-coding sequences and a fusion gene comprising a cell reprogramming factor or transcription factor selected from the group consisting of Oct4, Sox2, Klf4 and c-myc.

51 . The composition of claim 50 , wherein both the dendritic cell and the colorectal tumour cell are from the same or different subject.

52 . The composition claim 42 , wherein the undifferentiated colorectal stem-cell state is characterised by at least one of the following:
(a) loss of epithelial characteristics with reduction in E-cadherin expression, (b) gain of mesenchymal properties with differential expression of vimentin (VIM), fibronectin (FN1), vitronectin (VTN), N-cadherin (CDH2), snail (SNAI1), twist (TWIST1), zinc finger E-box-binding homeobox 1 (ZEB1), transforming growth factor beta 1 (TGFB1), slug (SNAI2) and SOX4; and (c) the expression of cancer stem cell markers selected from the group consisting of CD24, CD133, CD44, CD166, aldehyde dehydrogenase 1 (ALDH1A1), leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5), dipeptidyl peptidase 4 (DPP4), catenin beta-1 (CTNNB1), ATP-binding cassette sub-family G member 5 (ABCG5) and integrin beta-1 (ITGB1).

53 . The composition of claim 50 , wherein the undifferentiated colorectal stem-cell state is characterised by at least one of the following:
(a) loss of epithelial characteristics with reduction in E-cadherin expression, (b) gain of mesenchymal properties with differential expression of vimentin (VIM), fibronectin (FN1), vitronectin (VTN), N-cadherin (CDH2), snail (SNAI1), twist (TWIST1), zinc finger E-box-binding homeobox 1 (ZEB1), transforming growth factor beta 1 (TGFB1), slug (SNAI2) and SOX4; and (c) the expression of cancer stem cell markers selected from the group consisting of CD24, CD133, CD44, CD166, aldehyde dehydrogenase 1 (ALDH1A1), leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5), dipeptidyl peptidase 4 (DPP4), catenin beta-1 (CTNNB1), ATP-binding cassette sub-family G member 5 (ABCG5) and integrin beta-1 (ITGB1).

54 . A composition for pulsing a dendritic cell such that the dendritic cell is capable of inducing specific immune response of cytotoxic T lymphocytes against an in vitro colorectal cancer stem cell, said composition comprising at least one in vitro colorectal cancer stem cell-like cell, or fragments thereof, which has been enriched from an in vitro colorectal cancer cell that has been reprogrammed with a baculoviral vector into an undifferentiated colorectal stem-cell state, wherein the baculoviral vector comprises zinc-finger nuclease-coding sequences and a fusion gene comprising a cell reprogramming factor or transcription factor capable of reverting or reprogramming the differentiated colorectal tumour cell into the undifferentiated colorectal cancer stem-cell state.
The composition of claim 13 , wherein the inducing of the specific immune response comprises presenting antigens derived from said in vitro colorectal cancer stem cell to a population of T cells and inducing differentiation of said population of T cells into said cytotoxic T lymphocytes, wherein said cytotoxic T lymphocytes are specific against said antigens.

55 . The composition of claim 54 , wherein the inducing of the specific immune response comprises presenting antigens derived from said in vitro colorectal cancer stem cell to a population of T cells and inducing differentiation of said population of T cells into said cytotoxic T lymphocytes, wherein said cytotoxic T lymphocytes are specific against said antigens.

Assignees

• Agency Science Tech & Res
• Tessa Therapeutics Pte Ltd

Inventors

• Wang Shu
• Wu Chunxiao
• Zeng Jieming
• Purwanti Yovita Ida
• Khoo Andrew

Classifications

• C12N5/0696

  Artificially induced pluripotent stem cells, e.g. iPS · CPC title

• C12N2502/45

  Artificially induced pluripotent stem cells · CPC title

• C12N2502/30

  tumour cells · CPC title

• C12N2506/30

  from cancer cells, e.g. reversion of tumour cells · CPC title

• C12N2501/603

  Oct-3/4 · CPC title