U.S. patent application number 16/485488 was filed with the patent office on 2020-02-13 for method for generating t cells progenitors.
This patent application is currently assigned to Assistance Publique - Hopitaux de Paris. The applicant listed for this patent is Assistance Publique - Hopitaux de Paris, Fondation Imagine - Institut des Maladies Genetiques, Institut National de la Sante et de la Recherche Medicale, Universite Paris Descartes. Invention is credited to Isabelle Andre, Marina Cavazzana, Kuiying Ma, John Tchen.
Application Number | 20200046767 16/485488 |
Document ID | / |
Family ID | 58185453 |
Filed Date | 2020-02-13 |
![](/patent/app/20200046767/US20200046767A1-20200213-D00000.png)
![](/patent/app/20200046767/US20200046767A1-20200213-D00001.png)
![](/patent/app/20200046767/US20200046767A1-20200213-D00002.png)
![](/patent/app/20200046767/US20200046767A1-20200213-D00003.png)
![](/patent/app/20200046767/US20200046767A1-20200213-D00004.png)
![](/patent/app/20200046767/US20200046767A1-20200213-D00005.png)
![](/patent/app/20200046767/US20200046767A1-20200213-D00006.png)
![](/patent/app/20200046767/US20200046767A1-20200213-D00007.png)
![](/patent/app/20200046767/US20200046767A1-20200213-D00008.png)
United States Patent
Application |
20200046767 |
Kind Code |
A1 |
Andre; Isabelle ; et
al. |
February 13, 2020 |
METHOD FOR GENERATING T CELLS PROGENITORS
Abstract
The invention relates to an in vitro method to generate T cell
progenitors, comprising the step of culturing CD34+ cells in a
medium containing TNF-alpha and/or an antagonist of the Aryl
hydro-carbon/Dioxin receptor, in particular StemRegenin 1 (SR1), in
presence of a Notch ligand and optionally a fibronectin
fragment.
Inventors: |
Andre; Isabelle; (Issy Les
Moulineaux, FR) ; Cavazzana; Marina; (Paris, FR)
; Ma; Kuiying; (Paris, FR) ; Tchen; John;
(Pantin, FR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Assistance Publique - Hopitaux de Paris
Fondation Imagine - Institut des Maladies Genetiques
Universite Paris Descartes
Institut National de la Sante et de la Recherche Medicale |
Paris
Paris
Paris
Paris |
|
FR
FR
FR
FR |
|
|
Assignee: |
Assistance Publique - Hopitaux de
Paris
Paris
FR
Fondation Imagine - Institut des Maladies Genetiques
Paris
FR
Universite Paris Descartes
Paris
FR
Institut National de la Sante et de la Recherche Medicale
(INSERM)
Paris
FR
|
Family ID: |
58185453 |
Appl. No.: |
16/485488 |
Filed: |
February 12, 2018 |
PCT Filed: |
February 12, 2018 |
PCT NO: |
PCT/EP2018/053406 |
371 Date: |
August 13, 2019 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2501/125 20130101;
C12N 2501/145 20130101; C12N 5/0636 20130101; C12N 2501/26
20130101; C12N 2501/42 20130101; C12N 2501/2307 20130101; C12N
2506/11 20130101; A61K 35/17 20130101; A61K 35/00 20130101; C12N
2501/25 20130101; C12N 2533/52 20130101 |
International
Class: |
A61K 35/17 20060101
A61K035/17; C12N 5/0783 20060101 C12N005/0783 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 13, 2017 |
EP |
17305161.6 |
Claims
1.-20. (canceled)
21. An in vitro method for generating T cells precursors,
comprising the step of culturing CD34+ cells in a medium comprising
TNF-alpha and/or an antagonist of the Aryl hydrocarbon/Dioxin
receptor, in particular StemRegenin 1 (SR1), and in the presence of
an immobilized Notch ligand.
22. The in vitro method of claim 21, wherein the Notch ligand is
immobilized on the inner surface of a culture vessel or on the
surface of beads present in the culture medium.
23. The in vitro method of claim 21, wherein TNF-alpha and/or the
antagonist of the Aryl hydrocarbon/Dioxin receptor is present, in
the culture medium, from day 0 of the culture.
24. The in vitro method of claim 21, wherein TNF-alpha is present,
in the culture medium, at a concentration higher or equal to 3
ng/ml and/or wherein the antagonist of the Aryl hydrocarbon/Dioxin
receptor is present, in the culture medium, at a concentration
higher or equal to 30 ng/ml.
25. The in vitro method of claim 21, wherein the CD34+ cells have
been isolated from an adult donor.
26. The in vitro method of claim 21, wherein the cells are cultured
in the presence of TNF-alpha and/or SR1 for at most 10 days.
27. The in vitro method of claim 21, wherein the cells are cultured
in the presence of TNF-alpha and/or SR1 for between 3 and 7
days.
28. The in vitro method of claim 21, wherein the Notch ligand is
the soluble domain of the Delta-like-4 ligand, fused to an Fc
region of an IgG protein.
29. The in vitro method of claim 21, wherein the cells are also
exposed to a fibronectin fragment, wherein said fragment comprises
the RGDS and CS-1 patterns as well as a heparin-binding domain,
preferably immobilized on the inner surface of the culture vessel
or on beads.
30. The in vitro method of claim 21, wherein the cells are also
exposed to a fibronectin fragment, wherein the fibronectin fragment
is RETRONECTIN.RTM..
31. The in vitro method of claim 21, wherein the culture medium
also contains a vector intended for transfection or transduction of
the CD34+ cells, during at least some time of exposure of the CD34+
cells to the Notch ligand.
32. The in vitro method of claim 31, wherein the vector intended
for transfection or transduction of the CD34+ cells is a transgene
that codes for a Chimeric Antigen Receptor (CAR).
33. The in vitro method of claim 21, further comprising the step(s)
of: a. purifying the generated T cells progenitors, and b.
optionally conditioning the T cells progenitors in a pouch for
injection into a patient.
34. The in vitro method of claim 21, further comprising the step
of: exposing the cells to a vector intended for transfection or
transduction of CD34+ cells.
35. The in vitro method of claim 21, further comprising the step
of: exposing the cells to a vector or nucleic acid sequences
containing the element appropriate for gene editing.
36. A CD7+ T cell progenitors population wherein the proportion of
cells that are CD34- and CD1a- or CD34- and CD5-, or CD34- and
CD1a- and CD5- in this population is higher than 80%.
37. The CD7+ T cell progenitors population according to claim 36,
expressing a CAR.
38. A method for increasing the number of T cells in a subject in
need thereof, comprising administering progenitor T cells according
to claim 36 to a subject.
39. The method of claim 38, for treating lymphopenia.
40. The method of claim 38, for treating cancer, HIV infection,
partial thymectomy, autoimmune disease, and/or organ transplant.
Description
[0001] The invention relates to the field of cell therapy, in
particular of hematopoietic stem cells graft, transformed or not,
and more particularly of immune reconstitution after such
graft.
[0002] Graft of progenitor and Hematopoietic Stem/Progenitor Cell
(HSPC) is considered the best therapeutic option for the most
severe hereditary immune deficiencies, for many malignant
hemopathies, as well as for a number of solid tumors.
[0003] Currently, in allograft situations with partial HLA
incompatibility, the injections, to previously conditioned
recipients, of increasing doses of sorted CD34+ HSPC allows donor
transplantation with effective prevention of graft-versus-host
disease (GVH). Nevertheless, the differentiation of new T
lymphocytes from the injected CD34+ cells requires a minimum period
of 4 months and these T lymphocytes are in sufficient number to
play a protective role against infections only a few months after
their appearance.
[0004] This slowness of immune reconstitution leads to numerous
infectious complications especially viral, but also to relapses,
which influence the long-term prognosis of the grafted
patients.
[0005] In addition, other therapeutic protocols use a gene therapy
approach, namely an autograft of transduced HSPC, which has been
shown to be effective in the treatment of certain hereditary immune
deficiencies. The advantage of this strategy over HSPC CD34+
allogeneic transplantation is indisputable in terms of survival and
morbidity when no HLA-compatible donor is available. Nevertheless,
clinical experience has shown that, for some patients with severe
infections, reconstitution of the T lymphocyte compartment is slow
and never reaches normal levels of circulating T lymphocytes. The
morbidity and mortality associated with this particular context are
important.
[0006] Because of the high morbidity and mortality associated with
this type of transplant, the development of novel therapies to
reduce the immunodeficiency period after transplantation is fully
justified.
[0007] In particular, it is important to accelerate the generation
of T lymphocytes by the administration of precursors already
engaged in the T lymphocyte differentiation pathway (T cell
progenitors).
[0008] These T cell precursors are obtained from CD34+ HSPC
differentiation and have in particular the CD7+ marker, which is a
marker of differentiation in the T-cell pathway. They may also have
other markers. Awong et al (Blood 2009; 114: 972-982) described the
following precursors of T lymphocytes: early thymic progenitor
(ETP), which have markers (CD34+/CD45RA+/CD7+), precursor cells at
the proT1 stage (CD7++/CD5-), precursor cells at the proT2 stage
(CD7++/CD5+), and cells at the preT stage (CD7++/CD5+ CD1a+). The
HSPC acquire these markers in a successive way when passing from
one stage to the other, over the T cell development pathway. It is
further known that in humans, the CD1a antigen distinguishes the
passage from a very immature thymic progenitor to a progenitor
clearly engaged in the T-pathway (Cavazzana-Calvo et al,
MEDECINE/SCIENCES 2006; 22: 151-9).
[0009] A T-cell precursors transplant, concomitant with HSPC
grafting, would allow the rapid production of a mature and
functional T lymphocyte compartment and thus help prevent the risk
of severe infections by allowing the patient to benefit from some
immunity before complete reconstitution of the immune system.
[0010] Moreover, it is important to be able to use adult cells
rather than cord blood cells, since it is easier and cheaper to
obtain adult cells than cord blood cells and since adult cells are
more commonly used in allograft.
[0011] However, data published in the literature, obtained in
humans and mice, show intrinsic differences between fetal
hematopoietic cells (including cord blood) and adult cells. These
differences relate to survival, the ability to repair DNA damage,
proliferative capacity and potential to differentiate (see, for
example, Yuan et al., (2012 Mar. 9; 335 (6073): 1195-200), which
Indicate that adult bone marrow cells are less effective than fetal
cells in their potential to generate a variety of cell types:
Lansdorp et al (J Exp Med 1993 Sep. 1; 178 (3): 787-91); Szilvassy
et al (Blood, 2001 Oct. 1, 98 (7): 2108-15), Frassoni et al (Blood,
2003 Aug. 1, 102 (3): 1138-41), Liang et al. 106 (4): 1479-87), Six
et al (J Exp Med 2007 Dec. 24, 204 (13): 3085-93)).
[0012] WO 2016/055396 describes that it is possible to generate
T-cell precursors by culturing CD34+ cells in presence of an
immobilized ligand of Notch (in particular the soluble domain of
the Delta-like-ligand, fused to a Fc region of an IgG protein), and
of a fragment of a fibronectin, containing the RGDS (SEQ ID NO: 3,
Arginine-Glycine-Aspartate-Serine) and CS-1 domains, as well as the
heparin-binding domain (in particular in the presence of
Retronectin.RTM.). The Notch ligand used may be referred to as
DL4/Fc.
[0013] It is to be noted that this document discloses that presence
of both the immobilized ligand of Notch, and the fibronectin makes
it possible to increase generation of T lymphocytes progenitors
(CD7+ cells), which was already an improvement in the art, but that
the percentage of CD7+CD34- cells remains quite low, as shown in
FIG. 3 of WO 2016/055396. It is however interesting and
particularly important to increase this percentage, in order to be
able to administer a higher amount of precursor to the patient in
need thereof. On the other hand, it is also important that the
cells remains at a early stage of differentiation to be able to
provide a proper immunity.
[0014] It is reminded that Notch proteins are transmembrane
receptors that regulate the cellular response to a large number of
environmental signals. In mammals, four Notch (Notch 1-4) receptors
and five ligands (Delta-like-1, 3, and 4, Jagged1, Jagged2) have
been described (Weinmaster Curr Opin Genet Dev 2000: 10:
363-369).
[0015] The Delta-like-ligand 4 can be designated as: [0016] (ii)
Delta-like-ligand 4 (corresponding to the name of the DLL4 gene)
[0017] (iii) Delta-like-4 or Delta ligand 4 (abbreviation
DL-4).
[0018] In the present application, the Notch Delta-like-1 and
Delta-like-4 ligands may be designated respectively by DL1 and DL4
or by DL-1 and DL-4. The sequences of ligands DL-1 and DL-4 are
specified as SEQ ID NO: 1 and SEQ ID NO: 2, respectively.
[0019] Ohishi et al (BLOOD, vol. 98, no. 5, 2001, pp 1402-1407)
relates to the effect of Notch signaling on monocyte
differentiation into macrophages and dendritic cells. The monocyte
cells used in the experiments disclosed in this document were
either peripheral blood monocytes purified by negative selection,
or monocytes obtained after in vitro differentiation of CD34+ stem
cells. The cells used in this document have thus entered the
monocyte/macrophage/dendritic cells differentiation pathway, have
lost the CD34 marker (which is a hematopoietic stem cell marker)
and present the CD14 marker. These cells are cultured in presence
of the extracellular domain of Delta-1 and GM-CSF, TNF-alpha (which
is used to induce the differentiation of CD34+ cell-derived
CD1a-CD14+ cells into dendritic cells). This document this doesn't
uses CD34+ cells in presence of a Notch ligand and of TNF-alpha and
doesn't pertain to the generation of T-cell precursors (CD7+ T
lymphocytes precursors).
[0020] SHUKLA et al (NATURE METHODS, vol. 14, no. 5, 2017, 531-538)
and WO 2017/173551 disclose a method for generating progenitor T
cells from stem and/or progenitor cells comprising exposing the
stem and/or progenitor cells to Notch ligand Delta-like-4 (DL4) and
vascular adhesion molecule 1 (VCAM-1).
[0021] The method described in the present application allows the
generation and the increase (proliferation) of number of CD7+ T
lymphocyte precursors, from CD34+ stem cells, without using a cell
stroma (which can't be easily envisaged in a clinical context).
Furthermore, the method is particularly adapted when performed with
CD34+ cells issued from adult donors.
[0022] Furthermore, the cells obtained through the process as
herein disclosed, harbor the Bcl11b marker, which is important
transcriptional factor uniquely switched on since T-cell commitment
(Kueh et al, 2016, Nat Immunol. 2016 August; 17(8):956-65. doi:
10.1038/ni.3514).
[0023] The inventors have also shown absence of rearrangement of
the T cell receptors loci (either TCRbeta, TCRgamma or TCRdelta) in
the progenitors obtained through the process herein disclosed.
[0024] Moreover, a decrease in apoptosis markers was shown, for the
precursors herein obtained, as compared to the process disclosed in
WO 2016/055396.
[0025] In summary, the process herein disclosed makes it possible
to obtain a number of T cell precursors high enough to be
efficiently used for an adult receiving a stem cells
transplant.
[0026] This method may also be used to obtain transformed
(transduced) T cell precursors for gene therapy, when a vector
containing a gene of interest is used at some point during the
process herein disclosed.
[0027] The cells obtained by the process herein disclosed can be
obtained for adult CD34+ cells, and can be used for allogenic
grafts or autografts, even when cord blood cells are used for such
grafts.
[0028] It is to be noted that the method, although very efficient
for adult CD34+ cells, may also be used with cord blood CD34+
cells.
[0029] In a particular embodiment, the method herein disclosed thus
fasten T-cell generation in vivo after injection of T-cell
progenitors produced from HSPC in an in vitro culture system,
combining an immobilized fusion protein derived from the Notch
ligand, Delta-4, Retronectin.RTM. and a combination of
cytokines.
[0030] This system allows, within 7 days, the generation of T-cell
progenitors that are phenotypically and molecularly similar to
human thymic T-cell precursors. Furthermore, these T-cell
progenitors are able to give rise human mature and diverse T-cell
in NSG mice, with a faster kinetic as compared to HSPC.
[0031] The results herein reported were obtained from both cord
blood (CB) and adult (mobilized peripheral blood, mPB from adult
donors) HSPC.
[0032] The inventors have shown that the amount of T cell
precursors can be improved from CD34+ cells by exposing said CD34+
cells to a Notch ligand and in the presence of the soluble
TNF-alpha (Tumor Necrosis Factor Alpha, Uniprot P01375, RefSeq
NP_000585, SEQ ID NO: 8). Said exposure is made under conditions
suitable to generate progenitor T cells. Optionally, the cells are
also exposed to a fibronectin fragment containing an RDGS motif,
and/or a CS-1 motif and optionally a heparin binding domain.
Preferably, said fibronectin fragment contains an RDGS motif, a
CS-1 motif and a heparin binding domain.
[0033] TNF is primarily produced as a 233-amino acid-long type II
transmembrane protein arranged in stable homotrimers. The secreted
form of human TNF-alpha takes on a triangular pyramid shape, and
weighs around 17-kDa.
[0034] As disclosed in WO 2016/055396, it is possible to perform
the method herein disclosed, using a RGDS peptide and/or a CS-1
peptide in place of the fibronectin fragment. A combined use of the
RGDS and CS-1 peptides is preferred, in particular fused in the
same protein. Thus, the RGDS and/or CS-1 peptides may be present as
such in the culture medium or within a polypeptide or protein
present in the culture medium. When the culture medium only
contains the RGDS and/or CS-1 peptide as such, one can relate to
these as "free" peptide(s) in the culture medium if such peptides
are not immobilized on the inner surface of the culture vessel.
Indeed, the peptide(s) may be in solution or immobilized on the
inner surface of the vessel in which the CD34+ cells are exposed to
the immobilized Notch ligand.
[0035] However, as indicated above, it is preferred to use a
fragment of fibronectin, which contains the RGDS and CS-1 patterns,
as well as a heparin-binding domain. The fibronectin fragment may
be free in solution or immobilized on the inner surface of the
culture container.
[0036] The process is performed in vitro, in a container, such as a
cell culture plate (Petri dish, 24 well array or the like),
preferably with the Notch ligand immobilized on its inner surface.
The Notch ligand may, however, be immobilized on any other support
present in the culture medium, such as on the surface of beads (in
particular microbeads). Immobilization of the Notch ligand is
essentially intended to stabilize the ligand, in order to allow
activation of the Notch receptor of the CD34+ cells.
[0037] By "T cell progenitor", one intends to designate any cell
involved in the differentiation pathway to the T lymphoid pathway
from a CD34+ HSPC. This cell is therefore characterized in that it
expresses the CD7 marker, which is known to be one of the earliest
markers during the lymphopoiesis of the T cells. Depending on the
state of differentiation in the T lymphoid pathway, it can express
or not the CD34 marker (loss of CD34 during differentiation). Such
T cell progenitor may also express or not the CD5 marker.
[0038] Among the "T-cell progenitors" are those cells which can be
found in the post-natal thymus, i.e. early thymic progenitor (ETP)
(CD34+/CD45RA+/CD7+), proT1 cells (CD34+CD45RA+CD7++CD5-CD1a-),
proT2 cells (CD34+CD45RA+CD7++CD5+CD1a-) and preT cells
(CD34-CD7++/CD5+CD1a+). T-cell receptor (TCR) loci rearrange in a
highly ordered way (TCR.delta.-TCR.gamma.-TCR.beta.-TCR.alpha.). To
note, the first functional TCR rearrangements occurs at the
CD34-CD7++/CD5+CD1a+ preT cell stage (Dik et al. J Exp Med 2005;
201:1715-1723). T-cell progenitors are well known in the state of
the art. They are cited in particular by Reimann et al (STEM CELLS
2012; 30:1771-1780.) and by Awong et al (2009, op.cit.).
[0039] The term "RGDS peptide" is intended to designate any peptide
or protein that contains the RGDS pattern, so that it can bind
integrin VLA-5. Such peptide or protein can be tested for its
ability to bind VLA-5 integrin by methods known and reported in the
art. RGDS peptide binds to integrin VLA-5 (Very Late Antigen-5),
which is a dimer composed of CD49e (alpha5) and CD29 (beta1).
[0040] Heparin-binding domains are known in the art and present in
numerous proteins that bind to heparin. Their sequence is generally
XBBXBX or XBBBXXBX (B=acide amine basique; X=acide amine
hydropathique; Cardin and Weintraub, Arterioscler Thromb Vasc Biol.
1989; 9:21-32, SEQ ID NO: 4 et SEQ ID NO: 5).
[0041] Presence of such a heparin-binding domain is particularly
favorable when the CD34+ cells are exposed to a viral (especially a
retroviral) vector in order to transduce them and obtain T cell
progenitors expressing a transgene.
[0042] A CS-1 peptide or CS-1 pattern is a 25 amino acids peptide
(DELPQLVTLPHPNLHGPEILDVPST, SEQ ID NO: 6), described by Wayner et
al, 1989, J. Cell Biol. 109: 1321). This CS-1 pattern binds to the
VLA-4 (Very Late Antigen-4) receptor. This antigen is a dimer
integrin, composed of CD49d (alpha 4) and CD29 (beta 1).
[0043] In a particular embodiment, the fibronectin fragment is
present in the culture medium or immobilized on the inner (in
particular the bottom) wall of the container. Fibronectin is a
protein, which in its natural form is a v-shaped large dimer of 100
nm long and 460 kDa. The two monomers are connected by two
disulfide bridges at their C-terminus. The term "fibronectin" or
"fibronectin fragment" is understood to mean the natural
fibronectin protein (i.e. any isoform produced by alternative
splicing), but also a monomer of this protein, or a fragment of
this protein (but containing the peptide RGDS, as well as CS-1
peptide and heparin binding site).
[0044] A fibronectin which is particularly suitable for carrying
out the process herein disclosed is Retronectin.RTM.. This protein
corresponds to a fragment of a human fibronectin (CH-296 fragment,
Kimizuka et al., J Biochem., 1991 August 110 (2):284-91, Chono et
al., J Biochem 2001 September 130 (3):331-4) and contains the three
functional domains that are preferred for implementation of the
method (the cell-binding C domain containing the RGDS peptide, the
heparin-binding domain and the CS-1 sequence). This protein is sold
in particular by the company Takara Bio Inc. (Shiga, Japan).
[0045] In a particular embodiment, the fibronectin fragment is
immobilized (i.e. bound to a solid support and is not present free
in solution (although it is possible that certain elements may be
found in solution)). This solid support is preferably the bottom
wall of the container in which the process is carried out. However,
it is also possible to envisage binding the fibronectin fragment to
beads, such as polymer or magnetic beads (with a diameter generally
comprised between 1 and 5 .mu.m). The binding of the protein or
peptide to these beads may or may not be covalent. Methods for
attaching a protein or peptide to the beads are known in the art.
It is also possible to introduce the fibronectin fragment into a
semisolid medium, such as agar or gel.
[0046] When the fibronectin fragment is immobilized on the support
(in particular the bottom wall of the container in which the
process is performed), this immobilization may also be covalent or
not. In a preferred embodiment, this immobilization is carried out
non-covalently by allowing the fibronectin fragment to be absorbed
onto the glass or plastic composing the bottom wall of the
container.
[0047] In a particular embodiment, as seen above, the
differentiation of the CD34+ cells into T-cell progenitors is
carried out together with the transduction or transfection
(including Nucleofection.TM., a specific electroporation system
developed by Lonza) of the CD34+ cells by means of a vector (such
as a viral vector or a nucleic acid fragment such as a plasmid or
plasmidic RNA or DNA sequences) in order to introduce a gene of
interest (or a system for gene editing) in these cells. This means
that the cells exposed to the Notch ligand and the fibronectin
fragment, with TNF-alpha, are also exposed to a viral supernatant
for at least part of their time of exposure to the Notch ligand and
fibronectin fragment, with TNF-alpha.
[0048] The teachings of WO 2016/055396 with regards to the
operative conditions for performing cell transduction are expressly
applicable to the present method and are thus considered as being
expressly recited in this application.
[0049] One can cite, in particular: [0050] (iv) Exposure of the
cells to the Notch ligand, fibronectine fragment and TNF-alpha, for
some time (preferably more than 4 hours, more preferably more than
6 h, or more than 8 h or 10 h, but less than 36 h, more preferably
less than 30 h or less than 24 h) with appropriate cytokines known
in the art and disclosed in WO 2016/055396, and addition of the
viral supernatant for an appropriate duration (preferably more than
4 hours, more preferably more than 6 h, 8 h or 10 h, and preferably
less than 30 hours, more preferably less than 24 h, more preferably
around 16 h). [0051] (v) Second transduction if required as taught
in WO 2016/055396 [0052] (vi) Use of this protocol for autologous
hematopoietic stem cells grafts in gene therapy protocols, in order
for the transgene to add a protein that is absent or deficient in
the patient so as to bring a therapeutic benefit. [0053] (vii)
Usable transgenes: gene correcting immunodeficiencies (in
particular severe combined immunodeficiencies SCID or not, CID),
HIV, X-linked adrenoleukodystrophy, hemoglobinopathies, in
particular .beta.-thalassemy or sickle cell disease. One can also
use, as the transgene, a gene that codes for a Chimeric Antigen
Receptor (CAR), i.e. a cell surface protein that recognizes a cell
surface protein that is specifically expressed by cancer cells in
order to trigger immune response against the cancer cells through
the engineered CAR-T cells [0054] (viii) Preferred use of a viral
supernatant to allow insertion of the transgene within the cell
genome, using in particular lentiviruses known, and described in
the art. [0055] (ix) Introduction of the viral vector in the cell
culture medium after pre-activation of the CD34+ cells between 4
and 36 h, preferably between 6 and 24 h [0056] (x) Exposure of the
cells to the viral vector between 4 and 30 h, preferably between 12
and 24 h, more preferably around 16 h, and removal of the viral
vector (harvesting and washing the cells, and resuspending these
cells in presence of the Notch ligand, the fibronectin fragment and
the TNF-alpha).
[0057] As indicated above, in another embodiment, the CD34+ cells
are exposed to a system making it possible to perform gene editing.
Such systems are now widely known and described in the art and are
essentially based on nucleic acid double-break repair. Such genome
editing system may use a nuclease selected from the group
consisting of meganucleases, zinc finger nucleases (ZFNs),
transcription activator-like effector-based nucleases (TALEN), and
CRISPR-Cas nucleases. The fact that the CD34+ cells proliferate
during exposure to the elements as recited above, and thus in
presence of TNF-alpha, makes it possible to use these gene editing
systems that require proliferation of cells.
[0058] In a particular embodiment, the Notch ligand is the
Delta-like-1 protein (SEQ ID NO: 1) or a fragment thereof (soluble
domain).
[0059] In another and preferred embodiment, the Notch ligand is the
Delta-like-4 protein (SEQ ID NO: 2).
[0060] In a particular embodiment, said Notch ligand is a fusion
protein comprising the soluble domain of a natural Notch ligand
fused to an Fc region of an IgG protein. As known in the art, the
soluble domain of a Notch ligand represents the extracellular
portion of said ligand. Varnum-Finney et al (J Cell Sci., 2000
December; 113 Pt 23:4313-8) described a fusion protein of the
soluble part of DL-1 with an Fc portion of IGg1. Reimann et al (op
cit) described a fusion protein of the soluble part of DL-4 (amino
acids 1-526) with the Fc fragment of an IgG2b immunoglobulin. It is
thus preferred when the IgG protein is an IgG2. In a preferred
embodiment, the sequence of the Notch ligand used in the method
herein disclosed is SEQ ID NO: 7. A commercially available product
(Sino Biologicals) comprising the extracellular domain (Met 1-Pro
524) of human DLL4 (full-length DLL4 accession number NP_061947.1)
fused to the Fc region of human IgG 1 at the C-terminus is a DL4
protein is also suitable for use herein.
[0061] The culture medium used in the context of the method herein
disclosed is any medium adapted for culturing CD34+ cells and T
cells. Mention may in particular be made of .alpha.-MEM, DMEM, RPMI
1640, IMDM, BME, McCoy's 5A, StemSpan.TM., in particular SFII
(StemCell Technologies) media or Fischer's medium. StemSpan.TM.
SFII medium contains, in particular, Iscove's MDM, Bovine serum
albumin, Recombinant human insulin, Human transferrin
(iron-saturated), 2-Mercaptoethanol.
[0062] A suitable and preferred culture medium for carrying out the
process herein disclosed is the X-VIVO.TM. medium (Lonza, Basel,
Switzerland). This medium was used in particular by Jonuleit et al
(Eur J Immunol, 1997, 27, 12, 3135-42) and Luft et al (J Immunol,
1998, 161, 4, 1947-53).
[0063] Preferably, a basal medium is used (i.e., which a medium
that allows the growth of the cells without the need to add
supplements), in which, however, one preferably would add serum,
and/or growth factors and cytokines.
[0064] Thus, fetal bovine serum (FBS) or fetal calf serum (FCS),
autologous human serum or human AB serum is preferably added to the
basal culture medium. Preferably, this medium is supplemented with
at least 15% of fetal serum, more preferably at least 20%. The FBS
is particularly suitable for the implementation of the process. In
particular, it is preferred to use defined FBS. The defined FBS is
a high-quality serum which has been analyzed and filtered to avoid
the presence of viral particles. It is sold as such by many
suppliers, such as the HyClone.TM. Defined Fetal Bovine Serum (FBS)
of Thermo Scientific.TM..
[0065] In addition to TNF-alpha, the culture medium is also
preferably complemented with cytokines and growth factors. These
cytokines and growth factors are especially selected from the group
consisting of SCF (stem cell factor), thrombopoietin (TPO, also
called megakaryocyte growth and development factor, MGDF),
Flt3-Ligand (which is a growth factor Hematopoietic), interleukin 3
(IL-3), interleukin 7 (IL-7) and SCF (stem cell factor). In a
particular embodiment, the culture medium contains at least three,
preferably at least four of these cytokines or growth factors, in
addition to TNF-alpha.
[0066] In a preferred embodiment, and in particular for the
generation of T cell precursors that are not transduced with a
viral vector, at least or exactly three cytokines are added.
Preferably, these three cytokines are Interleukin-7 (IL-7), SCF
(Stem Cell Factor) and Flt-3 Ligand (hematopoietic growth
factor).
[0067] In another preferred embodiment, four cytokines, i.e. the
three cytokines mentioned above and TPO (thrombopoietin) are
added.
[0068] In another particular embodiment, and in particular for the
generation of T cell precursors transduced with a viral vector, the
nature of the cytokines and growth factors can be varied during the
implementation of the method.
[0069] Thus, IL-3, IL-7, SCF, TPO, and Flt3-L can be used in the
medium if the step of pre-activating the cells prior to addition of
the viral vector, and then supplement the medium only with IL-7,
SCF, TPO, and Flt3-L after the vector is removed.
[0070] The aforementioned cytokine and growth factor mixtures are
sufficient to induce the differentiation of CD34+ cells into T-cell
precursors and generally the culture medium comprises no other
cytokine or growth factor, except TNF-alpha, which, as described in
the examples, makes it possible to increase the number of T cell
precursors.
[0071] In the process herein foreseen, the total duration of
exposure of the CD34+ cells in the presence of the Notch ligand, of
the protein or peptide exhibiting the RGDS motif and of TNF-alpha,
is generally carried out for a time preferably of more than 3 days
and less than 10 days.
[0072] This exposure may vary depending on whether the cells are
transduced. Thus, an exposure time of three days may be sufficient
for adult stem cells not transduced, whereas it will generally be
longer for infantile stem cells (about 7 days) or when transduction
is performed.
[0073] CD34+ cells are obtained from a bone marrow puncture, from
umbilical cord blood or from peripheral blood from adult donors,
which have been mobilized particularly with G-CSF or any other
mobilizing agent known in the art. Methods for sorting CD34+ cells
are known in the art. In particular, magnetic beads having an
antibody recognizing CD34 on their surface can be used for this
purpose.
[0074] Preferably, the cell culture vessel is prepared by
immobilizing the Notch ligand and the fibronectin fragment on the
inner (preferably the lower) surface prior to exposing the CD34+
cells. Retronectin.RTM. or other fibronectin naturally adhere to
the plastic of the cell culture box (Petri dish or 24-well box, or
other). Similarly, if Notch ligand is used as a fusion protein with
the Fc fragment of an immunoglobulin, this Fc fragment also adheres
to the plastics. It is therefore sufficient to leave the container
in the presence of these compounds for a few hours in order to
obtain the appropriate coating. A method to coat such culture
container with the Notch ligand and the fibronectin fragment is
disclosed in details in WO 2016/055396 and can be applied for
implementing the method as herein disclosed.
[0075] It is reminded that, according to WO 2016/055396, around 75%
of DL-4 will adhere to the container surface when 5 .mu.g/ml is
used, the optimal dose being higher or equal to 1.25 .mu.g/ml and
preferably between 2.5 and 5 .mu.g/ml.
[0076] With regards to the fibronectin fragment, a concentration of
25 .mu.g/ml is particularly adapted (especially when
Retronectin.RTM. is used), although one can use other
concentrations (higher or lower).
[0077] In the implementation of the process herein disclosed, CD34+
cells are added at a concentration comprised between 10.sup.6 et
10.sup.7 cells/ml, in particular around 2.times.10.sup.6 CD34+
cells/ml in the culture vessel.
[0078] Depending on whether the cells are to be transduced and of
the CD34+ cells, one can use a plate of between 2 to 10 cm.sup.2
(i.e. a plate from 24 to 6 wells). When one uses a 24 wells plate,
one adds between 10.sup.4 and 10.sup.6 CD34+ cells in each well,
preferably from 2.times.10.sup.4 to 4.times.10.sup.5 CD34+ cells
per well. When a 6 well plate is used, one will add between
8.times.10.sup.4 and 2.times.10.sup.6 cells per well.
[0079] The quantity of cells to add is adapted by the person
skilled in the art, according to the container used.
[0080] The cells are placed in the well, in the selected basal
medium, supplemented with TNF-alpha, and preferentially
supplemented with growth factors and cytokines, as indicated
above.
[0081] The concentrations of cytokines or growth factors are
between 2 and 300 ng/ml. Preferably, the concentration is higher
than 40 ng/I and lower than 300 ng/ml or 200 ng/I, more preferably
around 100 ng/ml.
[0082] However, when one wishes to generate transduced T cell
progenitors, and when the cells are pre-activated before being
exposed to the viral vector, one can use higher concentrations
(around 300-400 ng/ml). In this embodiment, SCF and Flt3-L can be
used at des concentrations in the range of 300 ng/ml, TPO and IL-7
in the range of 100 ng/ml, and IL-3 at about 40 ng/ml.
[0083] TNF-alpha is preferably used at a concentration equal or
higher than 5 ng/ml. Indeed, as demonstrated in the examples, this
low concentration is enough to obtain the proliferation of the T
cell precursors, without modifying the differentiation pathway.
[0084] However, higher concentrations can also be used. In
particular, the TNF-alpha concentration can be as high as 300 ng/ml
or 200 ng/ml. An appropriate concentration is around 100 ng/ml.
However, other concentrations such as 10 ng/ml, 20 ng/ml or 50
ng/ml are also suitable.
[0085] The invention thus relates to an in vitro method for
generating T cells precursors comprising the step of culturing
CD34+ cells in a suitable medium comprising TNF-alpha and in the
presence of an immobilized Notch ligand, and optionally the
fibronectin fragment as disclosed above.
[0086] The invention thus relates to an in vitro method for
expanding T cells precursors comprising the step of culturing CD34+
cells in a suitable medium comprising TNF-alpha and in the presence
of an immobilized Notch ligand, and optionally the fibronectin
fragment as disclosed above.
[0087] Expanding the T cells precursors is intended to mean that
there are more T cell precursors than when the TNF-alpha is not
used, and/or that there are more T cell precursors than the number
of CD34+ cells introduced in the container.
[0088] In particular, the T cells precursors obtained by the above
methods are CD34-/CD7+/CD5- precursors.
[0089] Generally, the cells don't harbor the CD1a marker
either.
[0090] In a preferred embodiment, the T cells precursors obtained
by the above methods are CD34-/CD7+/CD1a- precursors.
[0091] This means that the population of T cell precursors obtained
by the present method express the CD7 marker, and that more than
80% of the cells expressing this marker have the above phenotype
(not expressing [0092] CD34 and CD1a, or [0093] CD34 and CD5, or
[0094] CD34 and CD1a and CD5),
[0095] as analyzed by flow cytometry. This phenotype is generally
obtained after 7 days of culture.
[0096] As indicated, the CD34+ cells are preferably not cord blood
cells. However, the method may also be used with and is applicable
to cord blood CD34+ cells, and it also leads to improved
results.
[0097] The method is particularly interesting when implemented with
CD34+ cells that have been isolated from an adult patient. Said
patient may be a healthy donor, or a donor with a disease, and
particularly for which the cells will be corrected by viral
transduction.
[0098] The cells may be cultured for more than 3 days, and
preferably for at least 5 days, more preferably for at least or
exactly 6 days, most preferably for at least or exactly 7 days,
although the duration of culture may last longer.
[0099] In the method herein disclosed, TNF-alpha is preferably
added to the culture medium from day 0 (i.e. at the beginning of
CD34+ cells culture) and shall remain present, in the culture
medium, for at least three days, and preferably during all the time
the cells are culture (i.e. preferably about 7 days).
[0100] When performing the method herein disclosed, it is possible
to further add StemRegenin 1 (SR1,
4-(2-(2-(Benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phen-
ol, CAS 1227633-49-10) in the culture medium already containing the
TNF-alpha.
[0101] The invention also relates to a method for obtaining T cells
progenitors, comprising the steps of [0102] a. performing the
method as disclosed above and [0103] b. purifying the generated T
cells progenitors thereby obtained.
[0104] Purification may be performed by washing the cells and
resuspending such in a basal medium.
[0105] This method may also comprise the step of conditioning the T
cells progenitors in a pouch for injection to a patient.
[0106] In this case, it is preferred when these cells are
reconditioned in a saline solution containing 5% HSA such as
Albunorm.TM. 5% 50 g/L (Octopharma, Lingolsheim, France).
[0107] These cells may also be frozen according to methods known in
the art.
[0108] The invention also relates to a pouch for intravenous
injection, containing a population of T cells progenitors
(susceptible to be obtained or as obtained by a method as described
above), among which the proportion of CD7+ cells in this population
is higher than 80%, more preferably higher than 85%.
[0109] The invention also relates to a pouch for intravenous
injection, containing a population of CD7+ T cells progenitors
(susceptible to be obtained or as obtained by a method as described
above), where the proportion of CD34- and CD5- cells (cells that
are CD7+ and both CD34- and CD5-) in this population is higher than
80%, more preferably higher than 85%.
[0110] The invention also relates to a pouch for intravenous
injection, containing a population of CD7+ T cells progenitors
(susceptible to be obtained or as obtained by a method as described
above), where the proportion of CD34- cells (cells that are CD7+
and CD34-) in this population is higher than 50%, more preferably
higher than 60%. Furthermore, at least 80%, and more preferably at
least 85% of the cells in this population are CD1a- cells.
[0111] The invention also relates to an in vitro method for
obtaining transformed T-cells progenitors, comprising the steps of
[0112] a. culturing CD34+ cells in a suitable medium comprising
TNF-alpha and in the presence of an immobilized Notch ligand [0113]
b. exposing the cells to a vector intended for transfection or
transduction of CD34+ cells.
[0114] As disclosed above, and after steps of washing and culturing
again the cells, transformed T-cells progenitors (expressing a
transgene, integrated within their genome) are obtained.
[0115] The invention also relates to an in vitro method for
obtaining modified T-cells progenitors, comprising the steps of
[0116] a. culturing CD34+ cells in a medium comprising TNF-alpha
and in the presence of an immobilized Notch ligand [0117] b.
exposing the cells to a vector or a nucleid acid sequence
containing the element appropriate for gene editing at least for
some time during cell culture.
[0118] Thereby T cells progenitors modified by gene editing are
obtained, after potential steps of washing and culturing.
[0119] The invention also relates to T cells progenitors, in
particular as obtained by the method herein disclosed, for their
use in an immunosuppressed patient, in particular for allowing
immune reconstitution in this patient and/or obtaining an immune
protection against infections in said patient, during a period of
some months (at least two months, preferably at least six
months).
[0120] In a particular embodiment, the patient is an
immunosuppressed patient. The reasons for the deficiency may be
multiple: hereditary immune deficiency, chemotherapy for leukemia,
conditioning, graft containing only stem cells, post-graft
treatment for prophylaxis of GVH (graft-versus-host disease), age
of patient, and complications such as infections.
[0121] In particular, the patient can be immunosuppressed due to
the depletion of its immune cells following therapy before
hematopoietic stem cells transplantation. In this embodiment, the
graft may be an allograft (in this case, the T cell progenitors are
preferably derived from a partially HLA-compatible donor), or an
autograft (in which case the T cell progenitors have preferentially
being transformed by a vector in order to express a gene and/or a
protein making it possible to correct a genetic defect in said
patient).
[0122] The T cell progenitors are preferably a population of CD7+
cells (i.e. a population where at least 75%, more preferably at
least 80% of cells in the population express the CD7 marker). This
population is also part of the invention. In particular, it is
susceptible to be obtained by a method herein disclosed. In a
specific embodiment, it is obtained by a method herein
disclosed.
[0123] More specifically, the T cell progenitors are preferably a
population of CD7+ cells (i.e. a population where at least 75%,
more preferably at least 80% of cells in the population express the
CD7 marker) where the proportion of CD34- and CD5- cells (cells
that are CD7+ and both CD34- and CD5-) in this population is higher
than 80%, more preferably higher than 85%. This population is also
part of the invention. In particular, it is susceptible to be
obtained by a method herein disclosed. In a specific embodiment, it
is obtained by a method herein disclosed.
[0124] In another embodiment, the T cell progenitors are preferably
a population of CD7+ cells (i.e. a population where at least 75%,
more preferably at least 80% of cells in the population express the
CD7 marker). In this population, more than 50%, more preferably
more than 60% of cells don't express the CD34 marker. In this
population at least 80%, more preferably at least 85% of cells
don't express the CD1a marker. The proportion of CD7+ CD1a- cells
in this population is preferably at least 80%. This population is
also part of the invention. In particular, it is susceptible to be
obtained by a method herein disclosed. In a specific embodiment, it
is obtained by a method herein disclosed.
[0125] In order to determine whether a cell is positive or not to a
surface marker (CD7, CD34, CD5 or CD1a), one shall use any method
known in the art, and in particular flow cytometry, after the cells
have been marked with fluorescent antibodies directed against the
surface antigen. The principle is that a signal will be emitted,
for each cell of the population, with a given intensity, and cells
are considered as positive for the antigen if the signal is higher
than a given threshold.
[0126] For CD34: a control population consisting of a population of
HPSC (such as one isolated from cord blood or from mobilized
peripheral blood) is used to determine the appropriate thresholds.
In this population of cells, the cells are CD34+ CD7- CD5-, or
CD34+ CD7- CD1a-. With this control, it is possible to determine
threshold for each antigen that will be used to determine whether
cells in another population are positive or not for these
antigens.
[0127] The control population generally will contain around 90% of
CD34+ CD7- CD5-, or of CD34+ CD7- CD1a- hematopoietic stem cells.
The threshold is the signal intensity level for which the cells of
the population can be sorted in a 90/10 proportion.
[0128] For CD7 (resp. CD5 or CD1a): a control population is used,
obtained by isolation of Peripheral Blood Mononuclear Cell (PBMC)
by any technique known in the art, and in particular a density
gradient technique such as Ficoll-Paque PLUS (GE Healthcare Life
Sciences). CD7 (resp. CD5 or CD1a) positive cells are isolated
using any technique known in the art such as the MACS.RTM.
technique (Magnetic Cell Isolation and Cell Separation, Miltenyi
Biotec) which uses magnetic beads with anti-CD7 (resp. anti.CD5 or
anti-CD1a) antibody. In this population, the cells will essentially
be CD7+ (resp. CD5+ or CD1a+), it is believed that the proportion
would be around 90/10.
[0129] The thresholds for assessing whether a cell is positive to a
surface antigen are determined using the control population for
this antigen, and would be the intensity for which more than 90% of
the cells of the control population have a higher signal
intensity.
[0130] Consequently, a cell is considered as being positive for an
antigen if the intensity signal for this antigen is higher than the
threshold as determined above, and negative for the antigen if the
intensity signal for this antigen is lower than the threshold as
determined above.
[0131] The fact that the vast majority of T cell progenitors in the
population don't express the CD1a marker may prove to be favorable,
as cells expressing this CD1a marker may not be very efficient to
reach the thymus and may thus be as efficient as CD1a- cells for in
vivo repopulation. Since the reconstitution potential of CD1a+
cells is uncertain, it is thus preferable to lower the amount of
such cells in the population.
[0132] The invention also relates to a method for treating an
immunosuppressed patient, in particular for the purpose of allowing
an immune reconstitution, at least temporarily, in this patient,
comprising the step of administering to said patient T-cell
progenitors, as described above.
[0133] This method may also include the step of obtaining such
progenitors by exposing CD34+ cells to a Notch ligand in the
presence of TNF-alpha (as described above) and preferably to a
protein or peptide having the RGDS motif and/or the CS1 motif, in
particular A fibronectin fragment as described above, under the
conditions mentioned above.
[0134] In particular, a therapeutically effective amount is
administered, that is to say of the order of 1 to 5.times.10.sup.6
of progenitors per kg, which makes it possible to provide the
patient with cells capable of playing a protective role with regard
to infections during a few months (of the order of about 6
months).
[0135] Preferably, this administration of T cell progenitors is
performed just prior to, just after or concomitantly with a
hematopoietic stem cell transplant in said patient. As seen above,
the injected cells can be transformed by a vector intended to allow
the correction of a genetic defect in said patient.
[0136] In another embodiment, the T cell progenitors are injected
to a patient that doesn't need a hematopoietic stem cell
transplant. Indeed, it is possible to use the transformed or
transduced T cells progenitors to treat some immunodeficiencies, in
which only T cells are affected, HIV or cancer patients (using the
progenitors modified to lead to CAR-T cells), without the need to
perform the immune system depletive chemotherapy.
[0137] It is to be noted that the teachings of the invention, as
disclosed with TNF-alpha are also applicable with the purine
derivative StemRegenin 1 (SR1, disclosed in Boitano et al, Science.
2010 Sep. 10; 329(5997):1345-8). Thus SR1 can be substituted to
TNF-alpha (i.e. used in place of TNF-alpha) to obtain T cell
progenitors from CD34+ cells, with a beginning of differentiation,
and expansion of the cells (increase in the number of cells). The
concentration of SR1 is preferably in the range of 750 nM (30
ng/ml), also higher concentration such as 1500 nM or 2500 nM (100
ng/ml), or even 5000 nM (200 ng/ml) or lower such as 500 nM (20
ng/ml) can also be envisaged. When used alone, but particularly in
combination with TNF-alpha, much lower concentrations can be used,
as low as 3 ng/ml, or 10 ng/ml. Suitable concentrations also
include 30 ng/ml or 100 ng/ml. Consequently, any concentration
higher than 3 ng/ml, or higher than 10 ng/ml is suitable to
increase the differentiation of the CD34+ cells to T-cells
progenitors.
[0138] SR1 is a ligand (an antagonist) of the Aryl
hydrocarbon/Dioxin receptor (AhR). Other AhR antagonists can be
used alone, or in combination with TNF-alpha, to promote
differentiation of CD34+ cells into the T-cell progenitor lineage.
One can cite resveratrol, omeprazole, Luteolin,
alpha-naphthoflavone, mexiletine, tranilast,
6,2',4'-Trimethoxyflavone, CH 223191
(1-Methyl-N-[2-methyl-4-[2-(2-methylphenyl)diazenyl]phenyl-1H-pyrazole-5--
carboxamide, CAS 301326-22-7). The amount of AhR antagonist is to
be determined on a case-by-case basis by one of skill in the art,
taking into consideration the IC50 of the antagonist (SR1 has an
IC50 of 127 nM, whereas CH 223191 has an IC50 of 30 nM) and the
fact that some products may have an agonist activity towards the
AhR when used at high concentrations (such as alpha-naphthoflavone)
or depending of the cellular context (such as omeprazole). In view
of the difference in IC50 between SR1 and CH 223191, it is foreseen
that an effective concentration of CH 223191 would be well below
the effective concentration of SR1, herein disclosed.
[0139] Consequently, the invention also relates to [0140] (i) an in
vitro method for generating T cells precursors, comprising the step
of culturing CD34+ cells in a medium comprising an antagonist of
the Aryl hydrocarbon/Dioxin receptor, in particular StemRegenin 1
(SR1) and in the presence of an immobilized Notch ligand. [0141]
(ii) The in vitro method as above, wherein the culture medium
further contains TNF-alpha. [0142] (iii) The in vitro method as
above, wherein the antagonist of the Aryl hydrocarbon/Dioxin
receptor, in particular SR1, is present, in the culture medium,
from day 0 of the culture. [0143] (iv) The in vitro method of any
of above, wherein the CD34+ cells have been isolated from an adult
donor, or from cord blood. [0144] (v) The in vitro method of any of
above, wherein the cells are cultured in presence of an antagonist
of the Aryl hydrocarbon/Dioxin receptor, in particular SR1, for at
most 10 days. [0145] (vi) The in vitro method of any of above,
wherein the cells are cultured in presence of the antagonist of the
Aryl hydrocarbon/Dioxin receptor, in particular SR1, between 3 and
7 days. [0146] (vii) The in vitro method of any of above, wherein
the antagonist of the Aryl hydrocarbon/Dioxin receptor, in
particular SR1, is added to the medium culture at a concentration
between 1 ng/ml and 300 ng/ml and preferably higher or equal to 1
ng/ml, or higher or equal to 3 ng/ml, or higher or equal to 10
ng/ml, and preferably lower than 200 ng/ml, or 150 ng/ml and
generally between 3 ng/ml and 100 ng/ml. [0147] (viii) The in vitro
method of any of above, wherein the Notch ligand is the soluble
domain of the Delta-like-4 ligand, fused to a Fc region of an IgG
protein, preferably an IgG2 protein. [0148] (ix) The in vitro
method of any of above, wherein the cells are also exposed to a
fibronectin fragment, wherein said fragment comprises the RGDS and
CS-1 patterns as well as a heparin-binding domain, preferably
immobilized on the inner surface of the culture vessel. [0149] (x)
The in vitro method of above, wherein the fibronectin fragment is
Retronectin.RTM., as disclosed above. [0150] (xi) The in vitro
method of any of above, wherein the culture medium also contains a
vector intended for transfection or transduction of the CD34+
cells, during at least some time of exposure of the CD34+ cells to
the Notch ligand. [0151] (xii) The in vitro method of any of above,
wherein the culture medium contains at least three, and preferably
all four, cytokines or growth factors chosen from the group
consisting of Interleukin 7 (IL-7), SCF (Stem Cells Factor),
thrombopoietin (TPO), and Flt3 ligand (FLT3L). [0152] (xiii) An (in
vitro) method for obtaining T cells progenitors, comprising the
steps of [0153] a. performing the method according to any of above
and [0154] b. purifying the generated T cells progenitors [0155] c.
optionally conditioning the T cells progenitors in a pouch for
injection to a patient. [0156] (xiv) An in vitro method for
obtaining transformed T-cells progenitors, comprising the steps of
[0157] a. culturing CD34+ cells in a medium comprising an
antagonist of the Aryl hydrocarbon/Dioxin receptor, in particular
SR1, and in the presence of an immobilized Notch ligand [0158] b.
exposing the cells to a vector intended for transfection or
transduction of CD34+ cells. [0159] (xv) An in vitro method for
obtaining modified T-cells progenitors, comprising the steps of
[0160] a. culturing CD34+ cells in a medium comprising an
antagonist of the Aryl hydrocarbon/Dioxin receptor, in particular
SR1, and in the presence of an immobilized Notch ligand [0161] b.
exposing the cells to a vector or nucleic acid sequences containing
the element appropriate for gene editing.
[0162] The invention also relates to a kit for performing any
method as indicated above, comprising: [0163] (i) a coating medium
containing a ligand of Notch (in particular the soluble domain of
the Delta-like-ligand, fused to a Fc region of an IgG protein, in
particular a IgG2 protein), and optionally a fibronectin fragment
[0164] (ii) a medium adapted for culturing (and/or expanding) CD34+
cells and T cells such as .alpha.-MEM, DMEM, RPMI 1640, IMDM, BME,
McCoy's 5A, StemSpan.TM. SFII (StemCell Technologies) media,
X-VIVO.TM. medium or Fischer's medium [0165] (iii) a progenitor
expansion medium containing TNF-alpha and preferably three
cytokines, in particular selected from SCF, TPO, Flt3L, and IL-7.
It is preferred when the progenitor expansion medium contains
TNF-alpha and all four cytokines SCF, TPO, Flt3L, and IL-7.
[0166] In another embodiment, the invention relates to a kit
comprising the same elements (i) and (ii) as indicated above, and a
progenitor expansion medium (iii) which contains an antagonist of
the Aryl hydrocarbon/Dioxin receptor, in particular SR1, and
preferably three cytokines, in particular selected from SCF, TPO,
Flt3L, and IL-7. It is preferred when the progenitor expansion
medium contains SR1 and all four cytokines SCF, TPO, Flt3L, and
IL-7.
[0167] In another embodiment, the progenitor expansion medium (iii)
contains TNF-alpha and an antagonist of the Aryl hydrocarbon/Dioxin
receptor, in particular SR1, and preferably three cytokines, in
particular selected from SCF, TPO, Flt3L, and IL-7. It is preferred
when the progenitor expansion medium contains TNF-alpha, SR1 and
all four cytokines SCF, TPO, Flt3L, and IL-7.
[0168] Such kit is particularly adapted and designed for performing
the methods herein disclosed.
[0169] The coating medium (i) is first used to coat the walls of a
culture vessel.
[0170] The medium (ii) is then used to culture CD34+ cells (either
obtained from cord blood or from mobilized peripheral blood, in
particular from an adult). Such medium is generally and preferably
used at a 1.times. concentration (i.e. it can be used without
dilution).
[0171] The medium (iii) is generally presented as a 10.times.
dilution (i.e. it has to be diluted in a medium (ii) for use. The
reconstituted medium from media (ii) and (iii) is then used to
promoter differentiation of the CD34+ cells to T-cell progenitors,
in the T-cell lineage, as disclosed above.
[0172] The invention also relates to a method for increasing the
number of T cells in a subject in need thereof, the method
comprising administering to the subject an effective number of
progenitor T cells as obtained by a method disclosed herein.
[0173] The invention also relates to progenitor T cells as obtained
by any method herein disclosed for their use for treating a subject
in need of an increased number of T cells.
[0174] In particular, the subject is a human.
[0175] In particular, the administered progenitor T cells are
autologous. In another embodiment, the administered progenitor T
cells are allogeneic.
[0176] In particular, the subject in need of the increased number
of T cells has a medical condition causing or resulting in
lymphopenia, in particular cancer, HIV infection, partial
thymectomy, autoimmune disease, and/or organ transplant.
DESCRIPTION OF THE FIGURES
[0177] FIG. 1: Total nucleated cells number obtained starting with
20000 CD34+ cells from cord blood (CB, FIG. 1.A) or mobilized
peripheral blood (mPB, FIG. 1.B), after 3 days (black bars) and 7
days (cumulative black and grey bars) of culture. NC: not
complemented; SR1: addition of StemRegenin 1 (750 nM); TNF-alpha:
addition of TNF-alpha (100 ng/ml); + (a), (b), (c): number of cells
observed when the complements are added from 0-3 days of culture
(a), 0-7 days of culture (b) or 4-7 days of culture (c).
[0178] FIG. 2: Number of CD7+ T-cell precursors obtained at day 7
starting with 20000 CD34+ cells from cord blood (CB, FIG. 2.A) or
mobilized peripheral blood (mPB, FIG. 2.B). Black bars: CD34+CD7+
cells; grey bars: CD34-CD7+ cells. (a), (b), (c): number of cells
observed when the complements are added from 0-3 days of culture
(a), 0-7 days of culture (b) or 4-7 days of culture (c).
[0179] FIG. 3: Expression of Bcl11b was analyzed on lived cells
after 7 days of culture obtained starting with CD34+ cells from
cord blood (CB) or mobilized peripheral blood (mPB) cultured in the
presence (grey bars) or absence (black bars) of TNF-alpha.
[0180] FIG. 4: Combined effect of TNF-alpha and the Notch ligand
DL4 on the number of CD7+ cells obtained at day 7 starting from
CD34+ cord blood cells (CB, black bars, left) or mobilized
peripheral blood (mPB, grey bars, right). (+/-means presence or
absence of DL4 or TNF-alpha).
[0181] FIG. 5: Frequency of myeloid cells at day 7 obtained
starting from CD34+ cells from cord blood (CB) or mobilized
peripheral blood (mPB) in presence (grey bars) or not (black bars)
of TNF-alpha (Mean.+-.SEM).
[0182] FIG. 6: Total CD7+ cells number obtained from day 3 to day 7
in a dose response assay of TNF-alpha, starting with CD34+ cells
from cord blood (CB, FIG. 6.A) or mobilized peripheral blood (mPB,
FIG. 6.B).
[0183] FIG. 7: Proportion of CD34-CD7+ cells (grey bars) vs
CD34+CD7+ cells (black bars) in a dose response assay of TNF-alpha
starting with CD34+ cells from cord blood (CB, FIG. 7.A) or
mobilized peripheral blood (mPB, FIG. 7.B)
[0184] FIG. 8: Proportion of cells in the different phases of the
cellular cycle. A. CB: cells differentiated from cord blood; B.
cells differentiated from mobilized peripheral blood cells. NC: non
complemented; TNF-alpha: cultured in presence of TNF-alpha (20
ng/ml); SR1: cultures in presence of SR1 (30 ng/ml)
[0185] FIG. 9: percentage (A) and total number (B) of CD5+CD7+
cells cultured starting with CD34+ cells from cord blood in
presence of TNF-alpha and/or SR1 at various concentrations, after 7
days of culture.
EXAMPLES
Example 1--Material and Methods
Human Cells
[0186] Cord blood samples not eligible for banking were used for
research purposes, following the provision of informed consent by
the child's mother. Mobilized Peripheral Blood (mPB) samples were
collected from healthy donors after G-CSF mobilization. Samples
were directly enriched for CD34+ cells. The informed consent was
given by each donor (Biotherapy Department, Necker Hospital,
Paris).
Exposure of CD34+ Progenitor Cells to Notch Ligand DL-4
[0187] CD34+ cells from human CB or mobilized peripheral blood
samples were cultured in 24-well plates or 6-well plates that had
been coated with recombinant human fibronection (RetroNectin.RTM.,
Clontech/Takara) and DL-4 (5 .mu.g/ml, PX'Therapeutics, Grenoble,
France). Coating was performed for 2 h at 37.degree. C., DL-4
coated wells were then blocked with bovine serum albumin 2% (BSA)
in phosphate-buffered saline (PBS) for 30 minutes at 37.degree. C.
and washed with PBS. Cultures were initiated at a concentration of
2.times.10.sup.4 cells/well or 1.times.10.sup.5 cells/well (for
24-well and 6-well plates respectively) in .alpha.-MEM medium
(Gibco, life Technology), supplemented with NaHCO.sub.3 (7.5%)
(Gibco, life Technology) and 20% defined fetal calf serum (Hyclone,
Thermo Fisher Scientific, Illkirch, France) and the recombinant
human cytokines interleukin-7 (IL-7), Flt3-ligand (Flt-3), stem
cell factor (SCF) and thrombopoietin (TPO) (all at 100 ng/ml and
all purchased from PeproTech Inc, Rocky Hill, N.J.) with or without
TNF-.alpha. (R&D Systems, US). After 3 days of culture, the
cells were half replaced by fresh medium. Cultured cells were
analyzed by fluorescence-activated cell sorting (FACS) after 3 and
7 days of culture on DL-4 respectively to exclude CD34-/CD7-
myeloid cells from subsequent analyses.
In Vitro T Cell Differentiation Assay on OP9/DL1 Cells
[0188] The T-lymphoid potential of native CD34+ CB cells and
TNF-.alpha. induced T cell progenitors generated by exposure to
DL-4 was assessed in OP9/DL-1 co-cultures, as previously described
(Six et al, Blood Cells Mol Dis. 2011 Jun. 15; 47(1):72-8 and Six J
Exp Med. 2007 Dec. 24; 204(13):3085-93).
Quantitative, Real-Time Polymerase Chain Reactions Using RT2
Profiler Array
[0189] CD7+ cells were sorted on Ariall after 7 days of culture.
Total RNA of sorted cell fractions from day 3 and day 7 was
isolated with the Rneasy Micro Kit (Qiagen, Courtaboeuf, France).
RT2 Profiler PCR arrays were performed following the protocol
detailed in the RT2 Profiler PCR Array Handbook (SA Biosciences,
Frederick Md.).
Flow Cytometry Analysis and Cell Sorting
[0190] Monoclonal antibodies against human CD34 (AC136), CD3
(BW264/56), CD45 (5B1) were purchased from Miltenyi Biotech
(Bergisch Gladbach, Germany), and CD4 (SK4), CD7 (M-T701), CD25
(M-A251), 7-aminoactinomycin D (7AAD) were from BD Biosciences (San
Jose, Calif.). Anti-human CD8 (RPAT8) was from Sony Biotechnology
(San Jose, USA). The Anti-human Ctip2 (Bcl11b) antibody was from
Abcam (Cambridge, UK).
[0191] Human cells were stained and analyzed using a Gallios
analyzer (Beckman Coulter, Krefeld, Germany). Cells from xenogenic
recipients were analysed on a MACSQuant.RTM. apparatus (Miltenyi
Biotech, Bergisch Gladbach, Germany). The data were analyzed using
FlowJo software (Treestar, Ashland, Oreg.) after gating on viable,
7AAD-negative cells. Cell subsets were sorted on an ARIA II
system.
Cell Proliferation Assays
[0192] For cell proliferation assays, CD34+ cells from CB and mPB
were labeled using the CellTrace.TM. CFSE kit (Life Technologies,
Carlsbad, Calif.) prior to culture with DL-4 and TNF-alpha (Life
Technologies). The cells' staining intensity was measured prior to
culture each day from day 3 to day 7. CFSE-positive cell were
analyzed on a Gallios cytometer (Beckman Coulter).
Cell Cycle Assays
[0193] For cell cycle analysis, cells were stained with
Hoechst33342 (Life technology) and Ki67-PC5 (BD Bioscience) after
fixed with Fixative reagent of PerFix-nc kit (Beckman Coulter) at
room temperature for 15 min, and added permeablizing reagent. The
data were analyzed using FlowJo software (version 10.2, Treestar,
Ashland, Oreg.) after gating on viable, 7AAD-negative cells.
Adoptive Transfer of In Vitro-Generated T-Cell Progenitors Derived
from Adult HSPCs into NSG Neonates
[0194] All experiments and procedures with animals were performed
in compliance with the French Ministry of Agriculture's regulations
on animal experiments. The injection of in vitro generated human
T-cell progenitors in NSG mice has been approved by the Ministry of
Higher Education and Research (APAFIS 2101-2015090411495178v4).
[0195] The NSG (NOD-Scid(IL2Rg.sup.null)) mice (obtained from the
Jackson Laboratory, Bar Harbor, Me., http://www.jax.org) were kept
in a pathogen-free facility. Progeny derived from mPB CD34+ HSPCs
in 7-day DL-4 cultures with or without TNF .alpha.
(3.times.10.sup.5 or 1.times.10.sup.6) were injected
intra-hepatically into NSG neonates (0-4 days old). Control mice
were injected with either 3.times.10.sup.5 non-cultured mPB CD34+
cells or 100 ul PBS.
[0196] Average engraftment levels of NSG mice were determined from
4 to 12 weeks post-transplant. Flow cytometry analysis was
performed on freshly cells collected from femur, thymus, peripheral
blood and spleen. Cells were treated with 1.times. red blood cell
lysis buffer (Biolegend, US) and washed before stained by
antibodies.
Analysis of T Cell Receptor Diversity
[0197] TCR gene rearrangement analysis was performed in duplicate
and on the two independently purified subsets (average is
shown).
[0198] TCR-.delta. quantification (D .delta.2-D .delta.3, D
.delta.2-J .delta.1, and D .delta.3-J .delta.1) was performed with
the listed sets of primers and probes.
[0199] The following were used for D .delta.2-D .delta.3
rearrangements:
TABLE-US-00001 D .delta.2, (SEQ ID No 9)
5'-CAAGGAAAGGGAAAAAGGAAGAA-3'; D .delta.3, (SEQ ID No 10)
5'-TTGCCCCTGCAGTTTTTGTAC-3'; and D'3 probe, (SEQ ID No 11)
5'-ATACGCACAGTGCTACAAAACCTACAGAGACCT-3'.
[0200] The following primers and probe were used for D .delta.2-J
.delta.1 rearrangements:
TABLE-US-00002 D .delta.2, (SEQ ID No 12)
5'-AGCGGGTGGTGATGGCAAAGT-3'; J .delta.1, (SEQ ID No 13)
5'-TTAGATGGAGGATGCCTTAACCTTA-3'; and J .delta.1 probe, (SEQ ID No
14) 5'-CCCGTGTGACTGTGGAACCAAGTAAGTAACTC-3'
[0201] The following were used for D .delta.3-J .delta.1
rearrangements:
TABLE-US-00003 D .delta.3, (SEQ ID No 15)
5'-GACTTGGAGAAAACATCTGGTTCTG-3';
[0202] J .delta.1 primer and J .delta.1 probe.
[0203] The analysis of TCR rearrangements by multiplex fluorescent
PCR was performed by separation of fluorochrome-labeled single
stand PCR products in a capillary sequencing polymer and detected
via automated laser scanning.
Apoptosis Assays
[0204] Cells were washed by cold PBS and resuspended in binding
buffer at a concentration of 1 million cells/ml. After adding 5 ul
Annexin V-PE (BD Bioscience) and 2 ul 7AAD, cells were incubated in
the dark at room temperature for 15 min. Subsequently, cells were
washed with 500 ul binding buffer and resuspended in 100 ul binding
buffer to be analyzed within 1 hour.
Example 2: Improvement on the Expansion and Differentiation of
T-Cell Precursors
[0205] When CD34+ cells are cultured with DL-4, FIG. 1 shows that
addition of TNF-alpha to the cell culture medium makes it possible
to multiply the total number of cells recovered at day 7 by 10
times as compared to culture without TNF-alpha, either when
starting with CD34+ cells issued from cord blood (FIG. 1.A) or from
PB (FIG. 1.B).
[0206] FIG. 2 shows that addition of TNF-alpha to the cell culture
medium makes it possible to multiply the number of CD7+ cells in by
20 to 40 times, either when starting with CD34+ cells issued from
cord blood (FIG. 2.A) or from PB (FIG. 2.B). The improvement is
especially high for the CD34-CD7+ cell population.
Example 3: Analysis of the Surface Markers of the T Cell
Progenitors
[0207] The surface markers present at the surface of the cells
obtained after 7 days of culture were determined by flow
cytometry.
TABLE-US-00004 Cord blood mPB CD34+ CD34- CD34+ CD34- CD34+ CD34-
CD34+ CD34- CD7+ CD7+ CD7- CD7- CD7+ CD7+ CD7- CD7- -TNF.alpha.
29.5 38.9 15.4 16.3 15.1 11.3 32.9 40.7 +TNF.alpha. 0.39 95.6 0.76
3.22 0.68 90.1 2.75 6.49
TABLE-US-00005 Cord blood mPB CD5+ CD5- CD5+ CD5- CD5+ CD5- CD5+
CD5- CD7+ CD7+ CD7- CD7- CD7+ CD7+ CD7- CD7- -TNF.alpha. 1.56 66.8
5.10.sup.-3 31.6 0.069 26.4 0.027 73.5 +TNF.alpha. 0.66 95.4 0.012
3.97 3.01 87.7 0.24 9.00
[0208] These tables show that addition of TNF-alpha leads to an
increase in the proportion of CD7+ cells, without really increasing
the proportion of CD5+ cells.
[0209] After 7 day culture, HSPCs differentiate into CD34-CD7+CD5-
T-cell precursors.
[0210] The surface markers present at the surface of the cells
obtained after 10 days of culture were also determined by flow
cytometry.
[0211] No expression of CD1a was found (data not shown).
[0212] The kinetics of modification of the surface markers was
studied and it was found that presence of TNF-alpha in the culture
medium increases the proportion of CD7+ from day 4 up to day 7
(data not shown).
Example 4: Rearrangement of T Cell Receptors
[0213] DL-4 T-cell precursors do not exhibit any signs of TCR
rearrangement with or without TNF-alpha or SR1 after 7 days of
culture (data not shown).
[0214] Specific analysis of rearrangement was performed:
Results of TCRdelta Rearrangements
[0215] Detection of D.delta.2-D.delta.3 rearrangements in CB-NC et
CB-SR1. No other TCRdelta rearrangements were detected. Results
were in accordance with RQ-PCR quantification
Results of TCRgamma Rearrangements
[0216] No TCRgamma rearrangements were detected.
Results of TCRbeta Rearrangements
[0217] No TCRbeta rearrangements were detected.
Example 5: T-Commitment of TNF .alpha. Induced T-Cell Precusors
[0218] Bcl11b is an important transcriptional factor uniquely
switched on since T-cell commitment and absolutely required for
T-cell differentiation.
[0219] Intracellular staining on T-cell precursors cultured with
TNF-alpha showed positive expression of Bcl11b for both CD34+ cells
issued from cord blood, and mPB. FIG. 3 shows that TNF-alpha
increases the proportion of total cells expressing Bcl11b
transcription factor. When cultured with TNF-alpha, the proportion
of Bcl11b expressing cells was increased.
Example 6: Differentiation on Other Lineages
[0220] Presence of other cell surface markers (CD14 and CD33)
specific of other lineages was assessed.
[0221] FIG. 5 shows that the culture in the presence of TNF-alpha
made such cells not detectable, whereas their proportion is less
than 22% when CD34+ cells are cultured without TNF-alpha.
Example 7: TNF-Alpha Reduces Apoptosis of the Cells
[0222] Apoptosis markers (7AAD and Annexin5) were studied.
TABLE-US-00006 7AAD +/- AnnexinV + apoptotic cells (%) CB mPB
-TNF.alpha. 1.66 10.56 +TNF.alpha. 0.28 0.96
[0223] This table shows that culture in presence of TNF-alpha
reduces the presence of the apoptosis markers. This is particularly
apparent for mPB.
Example 8: Dose Response Assay of TNF-Alpha
[0224] Various doses of TNF-alpha were used.
[0225] FIG. 6 shows that TNFa may increase CD7+ cell numbers during
culture, when the concentration is more than 10 ng/ml, either for
CB cells (FIG. 6.A) or mPB cells (FIG. 6.B).
[0226] Kinetics of the dose response showed that TNF-alpha
increases the T-cell differentiation after only 4 days of culture
in DL-4. There was no difference between the concentration 10, 50
and 100 ng/ml (not shown).
[0227] To determine the threshold of effective concentration,
analysis of lower concentrations (0.01-10 ng/ml) was performed.
[0228] The effect of TNF-alpha on CB and mPB on T-cell
differentiation (percentage of CD34-CD7+ cells) was found to be
concentration-dependent at low concentration (FIG. 7). The total
number of CD7+ T-cell precursors was not different from 5 ng/ml to
100 ng/ml.
Example 9: Proliferation Analysis During Culture
[0229] TNF-alpha was found to increase the proliferation of
CD34+CD7+ T-cell precursors since day 3 in DL-4 culture as compared
to culture conditions without TNF-alpha (data not shown).
Example 10: Synergy Between TNF-Alpha and the Notch Ligand
[0230] FIG. 4 shows that without DL4, both CB and mPB failed to
differentiate into CD7+ T-cell precursors. Even the complementation
of the medium with TNF-alpha couldn't rescue it.
[0231] When both TNF-alpha and the Notch ligand are present, the
effect observed is very high. It thus seems that there is a synergy
between these two compounds and that the effect of TNF-alpha on
T-cell differentiation is likely Notch dependant.
Example 11: Addition of TNF-Alpha Increases Proliferation of CD7+
Progenitors
[0232] After 7 days of culture in presence of TNF-alpha, CD34+CD7-,
CD34+CD7+ and CD34-CD7+ subsets were sorted, stained with CFSE
(Carboxyfluorescein succinimidyl ester) and the dilution of CFSE
(surrogate marker of cell proliferation) was followed from day 8 to
10.
[0233] Only CD34+CD7+ and CD34-CD7+ cells show increased
proliferation when cultured with TNF-alpha. (Data not shown)
Example 12: Cell Cycle Analysis
[0234] Analysis of the cell cycle was performed. It was observed
that more cells were released from G0 phase in presence of
TNF-alpha on both CB and mPB derived CD7+ progenitors (FIG. 8).
Example 13: Combination of SR1 and TNFa
[0235] SR1 accelerates T-cell differentiation as shown by the
presence of CD5+CD7+ cells at day 7. The number of CD5+CD7+ cells
is increased by the presence of both TNF-alpha and SR1 (FIG.
9).
Example 14: In Vivo Data
[0236] T-cell precursors induced in presence of TNF-alpha may
largely fasten the reconstitution of the T-lineage in vivo.
[0237] Indeed, 4 weeks post-transplantation, recipient mice
injected with mPB T-cell precursors produced in the presence of
TNF-alpha have larger thymus than mice injected with mPB T-cell
precursors produced without TNF-alpha. T-cell precursors induced in
presence of TNF-alpha can differentiate to activated
TCR.alpha..beta. T cells within 4 weeks in vivo. (Data not
shown)
[0238] In summary, addition of TNF-alpha from day 0 in the DL-4
culture system leads to an increase of T-cell progenitors (defined
by the surface expression of CD7) of 40 fold for mPB HSPC and 20
fold for CB HSPC at day 7.
[0239] The CD7+ T-cell progenitors generated from both CB and mPB
were mostly CD34- and were CD1a negative. Cells were also mostly
CD5 negative.
[0240] They expressed Bcl11b, which is important fine-turning
molecular for T-commitment and further T-cell differentiation.
[0241] They did not exhibit any signs of T-cell receptor
rearrangements.
[0242] Their phenotype and molecular characteristics were similar
to the one of the CD34-CD7+ T-cell progenitors obtained without
TNF-alpha.
[0243] Regarding the mechanisms involved in TNF-alpha action,
TNF-alpha decreases expression of apoptosis markers and increases
cell proliferation during the culture. It also inhibits myeloid
cell production.
[0244] The use of TNF-alpha in the DL4 culture system increase to a
huge extent the amounts of T-cell progenitors produced from both
human adult and cord blood HSPC. It may thus overcome the
difficulty to obtain large amounts of T-cell progenitors from adult
HSPC. It may also decrease the number of starting HSPC required in
future clinical trials and the quantity of GMP grade and other
reagents required, thus decreasing the costs of production of these
T-cell progenitors.
Sequence CWU 1
1
151723PRTArtificial SequenceHuman Delta -1 (soluble fraction 1-536)
G502 may be R 1Met Gly Ser Arg Cys Ala Leu Ala Leu Ala Val Leu Ser
Ala Leu Leu1 5 10 15Cys Gln Val Trp Ser Ser Gly Val Phe Glu Leu Lys
Leu Gln Glu Phe 20 25 30Val Asn Lys Lys Gly Leu Leu Gly Asn Arg Asn
Cys Cys Arg Gly Gly 35 40 45Ala Gly Pro Pro Pro Cys Ala Cys Arg Thr
Phe Phe Arg Val Cys Leu 50 55 60Lys His Tyr Gln Ala Ser Val Ser Pro
Glu Pro Pro Cys Thr Tyr Gly65 70 75 80Ser Ala Val Thr Pro Val Leu
Gly Val Asp Ser Phe Ser Leu Pro Asp 85 90 95Gly Gly Gly Ala Asp Ser
Ala Phe Ser Asn Pro Ile Arg Phe Pro Phe 100 105 110Gly Phe Thr Trp
Pro Gly Thr Phe Ser Leu Ile Ile Glu Ala Leu His 115 120 125Thr Asp
Ser Pro Asp Asp Leu Ala Thr Glu Asn Pro Glu Arg Leu Ile 130 135
140Ser Arg Leu Ala Thr Gln Arg His Leu Thr Val Gly Glu Glu Trp
Ser145 150 155 160Gln Asp Leu His Ser Ser Gly Arg Thr Asp Leu Lys
Tyr Ser Tyr Arg 165 170 175Phe Val Cys Asp Glu His Tyr Tyr Gly Glu
Gly Cys Ser Val Phe Cys 180 185 190Arg Pro Arg Asp Asp Ala Phe Gly
His Phe Thr Cys Gly Glu Arg Gly 195 200 205Glu Lys Val Cys Asn Pro
Gly Trp Lys Gly Pro Tyr Cys Thr Glu Pro 210 215 220Ile Cys Leu Pro
Gly Cys Asp Glu Gln His Gly Phe Cys Asp Lys Pro225 230 235 240Gly
Glu Cys Lys Cys Arg Val Gly Trp Gln Gly Arg Tyr Cys Asp Glu 245 250
255Cys Ile Arg Tyr Pro Gly Cys Leu His Gly Thr Cys Gln Gln Pro Trp
260 265 270Gln Cys Asn Cys Gln Glu Gly Trp Gly Gly Leu Phe Cys Asn
Gln Asp 275 280 285Leu Asn Tyr Cys Thr His His Lys Pro Cys Lys Asn
Gly Ala Thr Cys 290 295 300Thr Asn Thr Gly Gln Gly Ser Tyr Thr Cys
Ser Cys Arg Pro Gly Tyr305 310 315 320Thr Gly Ala Thr Cys Glu Leu
Gly Ile Asp Glu Cys Asp Pro Ser Pro 325 330 335Cys Lys Asn Gly Gly
Ser Cys Thr Asp Leu Glu Asn Ser Tyr Ser Cys 340 345 350Thr Cys Pro
Pro Gly Phe Tyr Gly Lys Ile Cys Glu Leu Ser Ala Met 355 360 365Thr
Cys Ala Asp Gly Pro Cys Phe Asn Gly Gly Arg Cys Ser Asp Ser 370 375
380Pro Asp Gly Gly Tyr Ser Cys Arg Cys Pro Val Gly Tyr Ser Gly
Phe385 390 395 400Asn Cys Glu Lys Lys Ile Asp Tyr Cys Ser Ser Ser
Pro Cys Ser Asn 405 410 415Gly Ala Lys Cys Val Asp Leu Gly Asp Ala
Tyr Leu Cys Arg Cys Gln 420 425 430Ala Gly Phe Ser Gly Arg His Cys
Asp Asp Asn Val Asp Asp Cys Ala 435 440 445Ser Ser Pro Cys Ala Asn
Gly Gly Thr Cys Arg Asp Gly Val Asn Asp 450 455 460Phe Ser Cys Thr
Cys Pro Pro Gly Tyr Thr Gly Arg Asn Cys Ser Ala465 470 475 480Pro
Val Ser Arg Cys Glu His Ala Pro Cys His Asn Gly Ala Thr Cys 485 490
495His Glu Arg Gly His Gly Tyr Val Cys Glu Cys Ala Arg Gly Tyr Gly
500 505 510Gly Pro Asn Cys Gln Phe Leu Leu Pro Glu Leu Pro Pro Gly
Pro Ala 515 520 525Val Val Asp Leu Thr Glu Lys Leu Glu Gly Gln Gly
Gly Pro Phe Pro 530 535 540Trp Val Ala Val Cys Ala Gly Val Ile Leu
Val Leu Met Leu Leu Leu545 550 555 560Gly Cys Ala Ala Val Val Val
Cys Val Arg Leu Arg Leu Gln Lys His 565 570 575Arg Pro Pro Ala Asp
Pro Cys Arg Gly Glu Thr Glu Thr Met Asn Asn 580 585 590Leu Ala Asn
Cys Gln Arg Glu Lys Asp Ile Ser Val Ser Ile Ile Gly 595 600 605Ala
Thr Gln Ile Lys Asn Thr Asn Lys Lys Ala Asp Phe His Gly Asp 610 615
620His Ser Ala Asp Lys Asn Gly Phe Lys Ala Arg Tyr Pro Ala Val
Asp625 630 635 640Tyr Asn Leu Val Gln Asp Leu Lys Gly Asp Asp Thr
Ala Val Arg Asp 645 650 655Ala His Ser Lys Arg Asp Thr Lys Cys Gln
Pro Gln Gly Ser Ser Gly 660 665 670Glu Glu Lys Gly Thr Pro Thr Thr
Leu Arg Gly Gly Glu Ala Ser Glu 675 680 685Arg Lys Arg Pro Asp Ser
Gly Cys Ser Thr Ser Lys Asp Thr Lys Tyr 690 695 700Gln Ser Val Tyr
Val Ile Ser Glu Glu Lys Asp Glu Cys Val Ile Ala705 710 715 720Thr
Glu Val2724PRTArtificial SequenceHuman delta-4 protein (soluble
fraction 1-526) 2Met Ala Ala Ala Ser Arg Ser Ala Ser Gly Trp Ala
Leu Leu Leu Leu1 5 10 15Val Ala Leu Trp Gln Gln Arg Ala Ala Gly Ser
Gly Val Phe Gln Leu 20 25 30Gln Leu Gln Glu Phe Ile Asn Glu Arg Gly
Val Leu Ala Ser Gly Arg 35 40 45Pro Cys Glu Pro Gly Cys Arg Thr Phe
Phe Arg Val Cys Leu Lys His 50 55 60Phe Gln Ala Val Val Ser Pro Gly
Pro Cys Thr Phe Gly Thr Val Ser65 70 75 80Thr Pro Val Leu Gly Thr
Asn Ser Phe Ala Val Arg Asp Asp Ser Ser 85 90 95Gly Gly Gly Arg Asn
Pro Leu Gln Leu Pro Phe Asn Phe Thr Trp Pro 100 105 110Gly Thr Phe
Ser Leu Ile Ile Glu Ala Trp His Ala Pro Gly Asp Asp 115 120 125Leu
Arg Pro Glu Ala Leu Pro Pro Asp Ala Leu Ile Ser Lys Ile Ala 130 135
140Ile Gln Gly Ser Leu Ala Val Gly Gln Asn Trp Leu Leu Asp Glu
Gln145 150 155 160Thr Ser Thr Leu Thr Arg Leu Arg Tyr Ser Tyr Arg
Val Ile Cys Ser 165 170 175Asp Asn Tyr Tyr Gly Asp Asn Cys Ser Arg
Leu Cys Lys Lys Arg Asn 180 185 190Asp His Phe Gly His Tyr Val Cys
Gln Pro Asp Gly Asn Leu Ser Cys 195 200 205Leu Pro Gly Trp Thr Gly
Glu Tyr Cys Gln Gln Pro Ile Cys Leu Ser 210 215 220Gly Cys His Glu
Gln Asn Gly Tyr Cys Ser Lys Pro Ala Glu Cys Leu225 230 235 240Cys
Arg Pro Gly Trp Gln Gly Arg Leu Cys Asn Glu Cys Ile Pro His 245 250
255Asn Gly Cys Arg His Gly Thr Cys Ser Thr Pro Trp Gln Cys Thr Cys
260 265 270Asp Glu Gly Trp Gly Gly Leu Phe Cys Asp Gln Asp Leu Asn
Tyr Cys 275 280 285Thr His His Ser Pro Cys Lys Asn Gly Ala Thr Cys
Ser Asn Ser Gly 290 295 300Gln Arg Ser Tyr Thr Cys Thr Cys Arg Pro
Gly Tyr Thr Gly Val Asp305 310 315 320Cys Glu Leu Glu Leu Ser Glu
Cys Asp Ser Asn Pro Cys Arg Asn Gly 325 330 335Gly Ser Cys Lys Asp
Gln Glu Asp Gly Tyr His Cys Leu Cys Pro Pro 340 345 350Gly Tyr Tyr
Gly Leu His Cys Glu His Ser Thr Leu Ser Cys Ala Asp 355 360 365Ser
Pro Cys Phe Asn Gly Gly Ser Cys Arg Glu Arg Asn Gln Gly Ala 370 375
380Asn Tyr Ala Cys Glu Cys Pro Pro Asn Phe Thr Gly Ser Asn Cys
Glu385 390 395 400Lys Lys Val Asp Arg Cys Thr Ser Asn Pro Cys Ala
Asn Gly Gly Gln 405 410 415Cys Leu Asn Arg Gly Pro Ser Arg Met Cys
Arg Cys Arg Pro Gly Phe 420 425 430Thr Gly Thr Tyr Cys Glu Leu His
Val Ser Asp Cys Ala Arg Asn Pro 435 440 445Cys Ala His Gly Gly Thr
Cys His Asp Leu Glu Asn Gly Leu Met Cys 450 455 460Thr Cys Pro Ala
Gly Phe Ser Gly Arg Arg Cys Glu Val Arg Thr Ser465 470 475 480Ile
Asp Ala Cys Ala Ser Ser Pro Cys Phe Asn Arg Ala Thr Cys Tyr 485 490
495Thr Asp Leu Ser Thr Asp Thr Phe Val Cys Asn Cys Pro Tyr Gly Phe
500 505 510Val Gly Ser Arg Cys Glu Phe Pro Val Gly Leu Pro Pro Ser
Phe Pro 515 520 525Trp Val Ala Val Ser Leu Gly Val Gly Leu Ala Val
Leu Leu Val Leu 530 535 540Leu Gly Met Val Ala Val Ala Val Arg Gln
Leu Arg Leu Arg Arg Pro545 550 555 560Asp Asp Gly Ser Arg Glu Ala
Met Asn Asn Leu Ser Asp Phe Gln Lys 565 570 575Asp Asn Leu Ile Pro
Ala Ala Gln Leu Lys Asn Thr Asn Gln Lys Lys 580 585 590Glu Leu Glu
Val Asp Cys Gly Leu Asp Lys Ser Asn Cys Gly Lys Gln 595 600 605Gln
Asn His Thr Leu Asp Tyr Asn Leu Ala Pro Gly Pro Leu Gly Arg 610 615
620Gly Thr Met Pro Gly Lys Phe Pro His Ser Asp Lys Ser Leu Gly
Glu625 630 635 640Lys Ala Pro Leu Arg Leu His Ser Glu Lys Pro Glu
Cys Arg Ile Ser 645 650 655Ala Ile Cys Ser Pro Arg Asp Ser Met Tyr
Gln Ser Val Cys Leu Ile 660 665 670Ser Glu Glu Arg Asn Glu Cys Val
Ile Ala Thr Glu Val Thr Pro Arg 675 680 685Leu Asp Leu Pro Ser Ala
Leu Phe Thr Leu His Pro Gly Trp Asp Val 690 695 700Phe His Met Gln
Arg Ala Ala Leu Arg Arg Arg Arg Glu Trp Gln Glu705 710 715 720Pro
Asp Arg Leu34PRTArtificial SequenceRGDS pattern 3Arg Gly Asp
Ser146PRTArtificial Sequenceheparin binding domain XBBXBX; B =
basic amino acid; X = hydropathic amino acidmisc_feature(1)..(1)Xaa
can be any naturally occurring amino acidmisc_feature(4)..(4)Xaa
can be any naturally occurring amino acidmisc_feature(6)..(6)Xaa
can be any naturally occurring amino acid 4Xaa Asx Asx Xaa Asx Xaa1
558PRTArtificial Sequenceheparin binding domain XBBBXXBX; B = basic
amino acid; X = hydropathic amino acidmisc_feature(1)..(1)Xaa can
be any naturally occurring amino acidmisc_feature(5)..(6)Xaa can be
any naturally occurring amino acidmisc_feature(8)..(8)Xaa can be
any naturally occurring amino acid 5Xaa Asx Asx Asx Xaa Xaa Asx
Xaa1 5625PRTArtificial SequenceCS-1 pattern 6Asp Glu Leu Pro Gln
Leu Val Thr Leu Pro His Pro Asn Leu His Gly1 5 10 15Pro Glu Ile Leu
Asp Val Pro Ser Thr 20 257754PRTArtificial Sequencefusion protein
human delta 4 - Fc receptor 7Met Ala Ala Ala Ser Arg Ser Ala Ser
Gly Trp Ala Leu Leu Leu Leu1 5 10 15Val Ala Leu Trp Gln Gln Arg Ala
Ala Gly Ser Gly Val Phe Gln Leu 20 25 30Gln Leu Gln Glu Phe Ile Asn
Glu Arg Gly Val Leu Ala Ser Gly Arg 35 40 45Pro Cys Glu Pro Gly Cys
Arg Thr Phe Phe Arg Val Cys Leu Lys His 50 55 60Phe Gln Ala Val Val
Ser Pro Gly Pro Cys Thr Phe Gly Thr Val Ser65 70 75 80Thr Pro Val
Leu Gly Thr Asn Ser Phe Ala Val Arg Asp Asp Ser Ser 85 90 95Gly Gly
Gly Arg Asn Pro Leu Gln Leu Pro Phe Asn Phe Thr Trp Pro 100 105
110Gly Thr Phe Ser Leu Ile Ile Glu Ala Trp His Ala Pro Gly Asp Asp
115 120 125Leu Arg Pro Glu Ala Leu Pro Pro Asp Ala Leu Ile Ser Lys
Ile Ala 130 135 140Ile Gln Gly Ser Leu Ala Val Gly Gln Asn Trp Leu
Leu Asp Glu Gln145 150 155 160Thr Ser Thr Leu Thr Arg Leu Arg Tyr
Ser Tyr Arg Val Ile Cys Ser 165 170 175Asp Asn Tyr Tyr Gly Asp Asn
Cys Ser Arg Leu Cys Lys Lys Arg Asn 180 185 190Asp His Phe Gly His
Tyr Val Cys Gln Pro Asp Gly Asn Leu Ser Cys 195 200 205Leu Pro Gly
Trp Thr Gly Glu Tyr Cys Gln Gln Pro Ile Cys Leu Ser 210 215 220Gly
Cys His Glu Gln Asn Gly Tyr Cys Ser Lys Pro Ala Glu Cys Leu225 230
235 240Cys Arg Pro Gly Trp Gln Gly Arg Leu Cys Asn Glu Cys Ile Pro
His 245 250 255Asn Gly Cys Arg His Gly Thr Cys Ser Thr Pro Trp Gln
Cys Thr Cys 260 265 270Asp Glu Gly Trp Gly Gly Leu Phe Cys Asp Gln
Asp Leu Asn Tyr Cys 275 280 285Thr His His Ser Pro Cys Lys Asn Gly
Ala Thr Cys Ser Asn Ser Gly 290 295 300Gln Arg Ser Tyr Thr Cys Thr
Cys Arg Pro Gly Tyr Thr Gly Val Asp305 310 315 320Cys Glu Leu Glu
Leu Ser Glu Cys Asp Ser Asn Pro Cys Arg Asn Gly 325 330 335Gly Ser
Cys Lys Asp Gln Glu Asp Gly Tyr His Cys Leu Cys Pro Pro 340 345
350Gly Tyr Tyr Gly Leu His Cys Glu His Ser Thr Leu Ser Cys Ala Asp
355 360 365Ser Pro Cys Phe Asn Gly Gly Ser Cys Arg Glu Arg Asn Gln
Gly Ala 370 375 380Asn Tyr Ala Cys Glu Cys Pro Pro Asn Phe Thr Gly
Ser Asn Cys Glu385 390 395 400Lys Lys Val Asp Arg Cys Thr Ser Asn
Pro Cys Ala Asn Gly Gly Gln 405 410 415Cys Leu Asn Arg Gly Pro Ser
Arg Met Cys Arg Cys Arg Pro Gly Phe 420 425 430Thr Gly Thr Tyr Cys
Glu Leu His Val Ser Asp Cys Ala Arg Asn Pro 435 440 445Cys Ala His
Gly Gly Thr Cys His Asp Leu Glu Asn Gly Leu Met Cys 450 455 460Thr
Cys Pro Ala Gly Phe Ser Gly Arg Arg Cys Glu Val Arg Thr Ser465 470
475 480Ile Asp Ala Cys Ala Ser Ser Pro Cys Phe Asn Arg Ala Thr Cys
Tyr 485 490 495Thr Asp Leu Ser Thr Asp Thr Phe Val Cys Asn Cys Pro
Tyr Gly Phe 500 505 510Val Gly Ser Arg Cys Glu Phe Pro Val Gly Leu
Pro Pro Ser Thr Met 515 520 525Val Arg Ser Val Glu Cys Pro Pro Cys
Pro Ala Pro Pro Val Ala Gly 530 535 540Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met Ile545 550 555 560Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 565 570 575Asp Pro
Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Met Glu Val His 580 585
590Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg
595 600 605Val Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn
Gly Lys 610 615 620Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro
Ala Pro Ile Glu625 630 635 640Lys Thr Ile Ser Lys Thr Lys Gly Gln
Pro Arg Glu Pro Gln Val Tyr 645 650 655Thr Leu Pro Pro Ser Arg Glu
Glu Met Thr Lys Asn Gln Val Ser Leu 660 665 670Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 675 680 685Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met 690 695 700Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp705 710
715 720Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
His 725 730 735Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser Pro 740 745 750Gly Lys8157PRTArtificial SequenceHuman
soluble TNF-alpha 8Val Arg Ser Ser Ser Arg Thr Pro Ser Asp Lys Pro
Val Ala His Val1 5 10 15Val Ala Asn Pro Gln Ala Glu Gly Gln Leu Gln
Trp Leu Asn Arg Arg 20 25 30Ala Asn Ala Leu Leu Ala Asn Gly Val Glu
Leu Arg Asp Asn Gln Leu 35 40 45Val Val Pro Ser Glu Gly Leu Tyr Leu
Ile Tyr Ser Gln Val Leu Phe 50 55 60Lys Gly Gln Gly Cys Pro Ser Thr
His Val Leu Leu Thr His Thr Ile65 70 75 80Ser Arg Ile Ala Val Ser
Tyr Gln Thr Lys Val Asn Leu Leu Ser Ala 85 90 95Ile Lys
Ser Pro Cys Gln Arg Glu Thr Pro Glu Gly Ala Glu Ala Lys 100 105
110Pro Trp Tyr Glu Pro Ile Tyr Leu Gly Gly Val Phe Gln Leu Glu Lys
115 120 125Gly Asp Arg Leu Ser Ala Glu Ile Asn Arg Pro Asp Tyr Leu
Asp Phe 130 135 140Ala Glu Ser Gly Gln Val Tyr Phe Gly Ile Ile Ala
Leu145 150 155923DNAArtificial SequencePrimer for detecting TCR
rearrangement 9caaggaaagg gaaaaaggaa gaa 231021DNAArtificial
SequencePrimer for detecting TCR rearrangement 10ttgcccctgc
agtttttgta c 211133DNAArtificial SequenceProbe for detecting TCR
rearrangement 11atacgcacag tgctacaaaa cctacagaga cct
331221DNAArtificial SequencePrimer for detecting TCR rearrangement
12agcgggtggt gatggcaaag t 211325DNAArtificial SequencePrimer for
detecting TCR rearrangement 13ttagatggag gatgccttaa cctta
251432DNAArtificial SequenceProbe for detecting TCR rearrangement
14cccgtgtgac tgtggaacca agtaagtaac tc 321525DNAArtificial
SequencePrimer for detecting TCR rearrangement 15gacttggaga
aaacatctgg ttctg 25
* * * * *
References