U.S. patent application number 16/245556 was filed with the patent office on 2019-05-16 for immuno-modulatory progenitor (imp) cell.
The applicant listed for this patent is CELL THERAPY LIMITED. Invention is credited to Martin John EVANS, Ajan REGINALD, Sabena SULTAN.
Application Number | 20190142869 16/245556 |
Document ID | / |
Family ID | 51266501 |
Filed Date | 2019-05-16 |
United States Patent
Application |
20190142869 |
Kind Code |
A1 |
REGINALD; Ajan ; et
al. |
May 16, 2019 |
IMMUNO-MODULATORY PROGENITOR (IMP) CELL
Abstract
The invention relates to immuno-modulatory progenitor (IMP)
cells and their use in therapy.
Inventors: |
REGINALD; Ajan; (Swansea,
GB) ; EVANS; Martin John; (Swansea, GB) ;
SULTAN; Sabena; (Swansea, GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
CELL THERAPY LIMITED |
Swansea |
|
GB |
|
|
Family ID: |
51266501 |
Appl. No.: |
16/245556 |
Filed: |
January 11, 2019 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
14904411 |
Jan 11, 2016 |
|
|
|
PCT/GB2015/051673 |
Jun 9, 2015 |
|
|
|
16245556 |
|
|
|
|
Current U.S.
Class: |
424/450 ;
435/325 |
Current CPC
Class: |
A61P 9/00 20180101; C12N
5/0663 20130101; C12N 5/0665 20130101; C12N 2500/02 20130101; A61P
1/02 20180101; A61P 37/02 20180101; A61P 19/00 20180101; A61P 9/10
20180101; C12N 2502/115 20130101; A61K 35/28 20130101; A61P 13/12
20180101; A61P 19/08 20180101; A61P 21/00 20180101; A61P 9/04
20180101; A61P 1/16 20180101; A61P 9/06 20180101; C12N 2500/84
20130101; C12N 5/0634 20130101; A61P 5/18 20180101; A61P 43/00
20180101; A61P 35/00 20180101; A61P 19/10 20180101; A61P 11/00
20180101; C12N 5/0633 20130101; A61P 19/04 20180101; A61P 35/04
20180101; C12N 2506/1353 20130101; A61P 19/02 20180101; A61P 29/00
20180101; C12N 2500/90 20130101 |
International
Class: |
A61K 35/28 20060101
A61K035/28; C12N 5/078 20060101 C12N005/078; C12N 5/071 20060101
C12N005/071 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 12, 2014 |
GB |
1410504.3 |
Claims
1. An isolated population of immuno-modulatory progenitor (IMP)
cells, wherein the IMP cells express detectable levels of MIC A/B,
CD304 (Neuropilin 1), CD178 (FAS ligand), CD289 (Toll-like receptor
9), CD363 (Sphingosine-1-phosphate receptor 1), CD99, CD181 (C-X-C
chemokine receptor type 1; CXCR1), epidermal growth factor receptor
(EGF-R), CXCR2, CD126, and HLA-ABC, and wherein at least 80% of the
population comprises the IMP cells.
2. An IMP cell population according to claim 1, wherein the
population comprises at least about 5.times.10.sup.5 cells.
3. An IMP cell population according to claim 1, wherein at least
85% of the population comprises the IMP cells.
4. An IMP cell population according to claim 1, wherein at least
90% of the population comprises the IMP cells.
5. An IMP cell population according to claim 1, wherein at least
97% of the population comprises the IMP cells.
6. An IMP cell population according to claim 1, wherein at least
98% of the population comprises the IMP cells.
7. An IMP cell population according to claim 1, wherein at least
99% of the population comprises the IMP cells.
8. An IMP cell population according to claim 1, wherein the IMP
cells secrete detectable levels of one or more of interleukin-6
(IL-6), IL-8, C-X-C motif chemokine 10 (CXCL10; interferon
gamma-induced protein 10; IP-10), Chemokine (C--C motif) ligand 2
(CCL2; monocyte chemotactic protein-1; MCP-1) and Chemokine (C--C
motif) ligand 5 (CCL5; regulated on activation, normal T cell
expressed and secreted; RANTES).
9. A pharmaceutical composition comprising (a) a population
according to claim 1 and (b) a pharmaceutically acceptable carrier
or diluent
Description
[0001] This application is a continuation of U.S. patent
application Ser. No. 14/904,411 filed Jan. 11, 2016, which is a
national phase application under 35 U.S.C. .sctn. 371 of
International Application No. PCT/GB2015/051673 filed Jun. 9, 2015,
which claims priority from GB Application No. 1410504.3 filed Jun.
12, 2014, which is incorporated herein by reference. The entire
contents of each of the above-referenced disclosures is
incorporated herein by reference without disclaimer.
FIELD OF THE INVENTION
[0002] The invention relates to immuno-modulatory progenitor (IMP)
cells and their use in therapy.
BACKGROUND TO THE INVENTION
[0003] Mesodermal cells are derived from a number of tissues and
act as the supportive structure for other cell types. Bone marrow
for instance is made of both haematopoietic and mesenchymal derived
cells. Two principle mesenchymal cell types have been previously
described and characterized, namely (i) mesenchymal stem cells
(MSCs) and their precursors and (ii) mesenchymal precursor cells
(MPCs) found in the bone marrow. Mesenchymal stem cells (MSCs) are
multipotent, adult stem cells. MSCs differentiate to form the
different specialised cells found in the skeletal tissues. For
example, they can differentiate into cartilage cells
(chondrocytes), bone cells (osteoblasts) and fat cells
(adipocytes).
[0004] MSCs are used in a variety of therapies, such as the
treatment of Age-related Macular Degeneration (AMD) and myocardial
infarct. Once administered to the patient, the MSCs typically
migrate (or home) to the damaged tissue and exert their therapeutic
effects through paracrine signaling and by promoting survival,
repair and regeneration of the neighbouring cells in the damaged
tissue.
[0005] Current therapies typically involve the infusion of a
mixture of MSC subtypes some of which do not migrate efficiently to
the tissue of interest. This necessitates the use of a high
cell-dose which can lead to off-target side effects and
volume-related side effects. MSCs are typically obtained from bone
marrow and so it is difficult to obtain large amounts.
SUMMARY OF THE INVENTION
[0006] This invention relates to a novel cell type that has not
been previously identified or isolated, the immuno-modulatory
progenitor cell. This IMP cell is quite distinct and different to
both MSCs and MPCs in its composition, function and characteristics
which impart an enhanced immuno-modulatory capacity.
[0007] The inventors have surprisingly identified a new
immuno-modulatory progenitor (IMP) cell having a specific marker
expression pattern. In particular, the IMP cell expresses MIC A/B,
CD304 (Neuropilin 1), CD178 (FAS ligand), CD289 (Toll-like receptor
9), CD363 (Sphingosine-1-phosphate receptor 1), CD99, CD181 (C-X-C
chemokine receptor type 1; CXCR1), epidermal growth factor receptor
(EGF-R), CXCR2 and CD126, The IMP cell expresses significantly
greater amounts of these markers than a mesencymal stem cell (MSC).
The IMP cells of the invention can be isolated from mononuclear
cells (MCs), such as peripheral blood MCs. The IMP cells are
capable of efficiently migrating to and repairing damaged tissues.
In particular, they are capable of homing, adherence,
transmigration, proliferation, angiogenic effects and paracrine
signalling.
[0008] Accordingly, the invention provides an immuno-modulatory
progenitor (IMP) cell, wherein the cell expresses detectable levels
of MIC A/B, CD304 (Neuropilin 1), CD178 (FAS ligand), CD289
(Toll-like receptor 9), CD363 (Sphingosine-1-phosphate receptor 1),
CD99, CD181 (C-X-C chemokine receptor type 1; CXCR1), epidermal
growth factor receptor (EGF-R), CXCR2 and CD126.
[0009] The invention also provides: [0010] a population of two or
more IMP cells of the invention; [0011] a population of
immuno-modulatory progenitor (IMP) cells, wherein [0012] (i) at
least 90% of the cells in the population express detectable levels
of MIC A/B, [0013] (ii) at least 60% of the cells in the population
express detectable levels of CD304 (Neuropilin 1), [0014] (iii) at
least 45% of the cells in the population express detectable levels
of CD178 (FAS ligand), [0015] (iv) at least 10% of the cells in the
population express detectable levels of CD289 (Toll-like receptor
9), [0016] (v) at least 15% of the population express detectable
levels of CD363 (Sphingosine-1-phosphate receptor 1), [0017] (vi)
at least 20% of the cells in the population express detectable
levels of CD99, [0018] (vii) at least 80% of the cells in the
population express detectable levels of CD181 (C-X-C chemokine
receptor type 1; CXCR1), [0019] (viii) at least 30% of the cells in
the population express detectable levels of epidermal growth factor
receptor (EGF-R), [0020] (xi) at least 60% of the cells in the
population express detectable levels of CXCR2 and [0021] (x) at
least 5% of the cells in the population express detectable levels
of CD126; [0022] a pharmaceutical composition comprising (a) an IMP
cell of the invention or a population of the invention and (b) a
pharmaceutically acceptable carrier or diluent, one or more
liposomes and/or one or more microbubbles; [0023] a method of
producing a population of IMP cells of the invention, comprising
(a) culturing mononuclear cells (MCs) under conditions which induce
the MCs to differentiate into IMP cells and (b) harvesting and
culturing those IMP cells which have an expression pattern as
defined above and thereby producing a population of the invention;
[0024] a method of repairing a damaged tissue in a patient,
comprising administering to the patient a population of the
invention or a pharmaceutical composition of the invention, wherein
the population or composition comprises a therapeutically effective
number of cells, and thereby treating the damaged tissue in the
patient; [0025] a population of the invention or a pharmaceutical
composition of the invention for use in a method of repairing a
damaged tissue in a patient; and [0026] a population of the
invention or a pharmaceutical composition of the invention for use
in a method of treating a cardiac, bone, cartilage, tendon,
ligament, liver, kidney or lung injury or disease in a patient.
DETAILED DESCRIPTION OF THE INVENTION
[0027] It is to be understood that different applications of the
disclosed products and methods may be tailored to the specific
needs in the art. It is also to be understood that the terminology
used herein is for the purpose of describing particular embodiments
of the invention only, and is not intended to be limiting.
[0028] In addition, as used in this specification and the appended
claims, the singular forms "a", "an", and "the" include plural
referents unless the content clearly dictates otherwise. Thus, for
example, reference to "a cell" includes "cells", reference to "a
tissue" includes two or more such tissues, reference to "a patient"
includes two or more such patients, and the like.
[0029] All publications, patents and patent applications cited
herein, whether supra or infra, are hereby incorporated by
reference in their entirety.
IMP Cell of the Invention
[0030] The present invention provides an immuno-modulatory
progenitor (IMP) cell. The IMP cell expresses detectable levels of
MIC A/B, CD304 (Neuropilin 1), CD178 (FAS ligand), CD289 (Toll-like
receptor 9), CD363 (Sphingosine-1-phosphate receptor 1), CD99,
CD181 (C-X-C chemokine receptor type 1; CXCR1), epidermal growth
factor receptor (EGF-R), CXCR2 and CD126.
[0031] MIC allows adaptation of cells and their immuno-behaviour in
an inflammatory context by decreasing their susceptibility to NK
killing.
[0032] CD304 (alternate name Neuropilin 1) is a co-receptor for
vascular endothelial growth factor (VEGF) and has roles in
angiogenesis, cell survival, migration and invasion.
[0033] CD178 (alternate name FAS ligand) maintains cell phenotype
and controls differentiation. It is also capable of inducing
proliferation of cells. Although FAS ligand is known primarily in
apoptotic signalling, it has been shown that FAS and FAS ligand
expressing cells are resistant to FAS-induced apoptosis.
[0034] CD289 (alternate name Toll-like receptor 9) is involved in
the modulation of immune responses and may facilitate cell
migration towards a target tissue.
[0035] Sustained activation of CD363 (alternate name is
Sphingosine-1-phosphate receptor 1) has resulted in increased
engraftment of cells in-vivo. CD363 also promotes angiogenesis,
modulates cell homing, modulates trafficking and migration of cells
and regulates chemotaxis.
[0036] CD99 is involved in cell adhesion and transmigration.
[0037] There are two classes of interleukin-8 (IL-8) receptors,
CXCR1 (or CD181) and CXCR2. Both receptors bind IL-8 with high
affinity, in contrast to the other CXC chemokines. Functionally,
CXCR1 and CXCR2 have been shown to play significant roles in
proliferation, migration, invasion and angiogenesis. Damaged
tissues release a variety of soluble inflammatory factors, such as
macrophage migration inhibitory factor (MIF) and interleukin-8, and
these factors may attract the IMP cells of the invention (and other
inflammatory cells) to the damaged tissue though binding to binding
CXCR1 and/or CXCR2.
[0038] EGF-R is involved in cell migration, adhesion and
proliferation.
[0039] CD126 (alternate name is IL-6R1) increases
immune-privilege
[0040] The IMP cells of the invention have numerous advantages. The
key advantages will be summarized here. However, further advantages
will become apparent from the discussion below.
[0041] The IMP cells of the invention may advantageously be used to
repair damaged tissues in patients. The IMP cells are capable of
efficiently migrating (or homing) to a damaged tissue and exerting
anti-inflammatory effects in the tissue. This is discussed in more
detail below. One of the most important abilities of the IMP cells
is to migrate (or home) to injured sites, which involves
chemotaxis. This is based on chemokine-signalling and utilises
mechanisms such as rolling, adhesion and transmigration. The
anti-inflammatory effects of the IMP cells promote survival, repair
and regeneration of the neighbouring cells in the damaged tissue.
The cells are also able to exert paracrine effects such as the
secretion of angiogenic, chemotactic and anti-apoptotic factors.
This is also discussed in more detail below.
[0042] As discussed in more detail below, the IMP cells are
produced from mononuclear cells (MCs), such as peripheral MCs,
taken from an individual, such as a human individual. Since the IMP
cells are produced from MCs, they may be produced easily (such as
from peripheral blood) and may be autologous for the patient to be
treated and thereby avoid the risk of immunological rejection by
the patient.
[0043] It is possible, in principle, to produce an unlimited number
of IMP cells from a single individual, since various samples of MCs
(i.e. various samples of blood) may be obtained. It is certainly
possible to produce very large numbers of IMP cells from a single
individual. The IMP cells of the invention can therefore be made in
large numbers.
[0044] The IMP cells of the invention are produced in clinically
relevant conditions, for instance in the absence of trace amounts
of endotoxins and other environmental contaminants, as well as
animal products such as fetal calf serum. This makes the IMP cells
of the invention particularly suitable for administration to
patients.
[0045] Since the IMP cells of the invention are produced from MCs,
they are substantially homologous and may be autologous. They also
avoid donor-to-donor variation, which frequently occurs with MSCs.
Numerous populations of IMP cells of the invention can be produced
from a single sample taken from the patient before any other
therapy, such as chemotherapy or radiotherapy, has begun.
Therefore, the IMP cells of the invention can avoid any of the
detrimental effects of those treatments.
[0046] The IMP cells of the invention can be made quickly. IMP
cells can be produced from MCs in less than 30 days, such as in
about 22 days.
[0047] The production of IMP cells from MCs avoids the moral and
ethical implications involved with using mesenchymal stem cells
MSCs derived from human embryonic stem cells (hESCs).
[0048] The IMP cells of the invention are typically produced from
human MCs. The IMP cells of the invention are therefore typically
human. Alternatively, the IMP cells may be produced from MCs from
other animals or mammals, for instance from commercially farmed
animals, such as horses, cattle, sheep or pigs, from laboratory
animals, such as mice or rats, or from pets, such as cats, dogs,
rabbits or guinea pigs.
[0049] The IMP cells of the invention can be identified as
immunomodulatory progenitor cells using standard methods known in
the art, including expression of lineage restricted markers,
structural and functional characteristics. The IMP cells will
express detectable levels of cell surface markers known to be
characteristic of IMPs. These are discussed below.
[0050] The IMP cells of the invention are capable of successfully
completing differentiation assays in vitro to confirm that they are
of mesodermal lineage. Such assays include, but are not limited to,
adipogenic differentiation assays, osteogenic differentiation
assays and neurogenic differentiation assays (Zaim M et al Ann
Hematol. 2012 August; 91(8):1175-86).
[0051] The IMP cells of the invention are not stem cells. In
particular, they are not MSCs. They are terminally differentiated.
Although they can be forced under the right conditions in vitro to
differentiating, for instance into cartilage or bone cells, they
typically do not differentiate in vivo. The IMP cells of the
invention have their effects by migrating to the damaged tissue and
exerting paracrine signalling in the damaged tissue. In particular,
the IMP cells are preferably capable of inducing anti-flammatory
effects in the damaged tissue. This is discussed in more detail
below.
[0052] The IMP cells of the invention are typically characterised
by a spindle-shaped morphology. The IMP cells are typically
fibroblast-like, i.e. they have a small cell body with a few cell
processes that are long and thin. The cells are typically from
about 10 to about 20 .mu.m in diameter.
[0053] The IMP cells of the invention are distinguished from known
cells, including MSCs, via their marker expression pattern. The
IMPs express detectable levels of MIC A/B, CD304 (Neuropilin 1),
CD178 (FAS ligand), CD289 (Toll-like receptor 9), CD363
(Sphingosine-1-phosphate receptor 1), CD99, CD181 (C-X-C chemokine
receptor type 1; CXCR1), epidermal growth factor receptor (EGF-R),
CXCR2 and CD126. The IMPs preferably express an increased amount of
these markers compared with MSCs. This can be determined by
comparing the expression level/amount of the markers in an IMP of
the invention with the expression level/amount in an MSC using the
same technique under the same conditions. Suitable MSCs are
commercially available. The MSC used for comparison is preferably a
human MSC. Human MSCs are commercially available from
Mesoblast.RTM. Ltd, Osiris Therapeutics.RTM. Inc. or Lonza.RTM..
The human MSC is preferably obtained from Lonza.RTM.. Such cells
were used for the comparison in the Example. The MSC may be derived
from any of the animals or mammals discussed above.
[0054] The IMP cells preferably express an increased amount of one
or more of MIC A/B, CD304 (Neuropilin 1), CD178 (FAS ligand), CD289
(Toll-like receptor 9), CD363 (Sphingosine-1-phosphate receptor 1),
CD99, CD181 (C-X-C chemokine receptor type 1; CXCR1), epidermal
growth factor receptor (EGF-R), CXCR2 and CD126 compared with a
MSC. The IMP cells preferably express an increased amount of all of
the ten markers compared with a MSC.
[0055] Standard methods known in the art may be used to determine
the detectable expression or increased expression of various
markers discussed above (and below). Suitable methods include, but
are not limited to, immunocytochemistry, immunoassays, flow
cytometry, such as fluorescence activated cells sorting (FACS), and
polymerase chain reaction (PCR), such as reverse transcription PCR
(RT-PCR). Suitable immunoassays include, but are not limited to,
Western blotting, enzyme-linked immunoassays (ELISA), enzyme-linked
immunosorbent spot assays (ELISPOT assays), enzyme multiplied
immunoassay techniques, radioallergosorbent (RAST) tests,
radioimmunoassays, radiobinding assays and immunofluorescence.
Western blotting, ELISAs and RT-PCR are all quantitative and so can
be used to measure the level of expression of the various markers
if present. The use of high-throughput FACS (HT-FACS) is disclosed
in the Example. The expression or increased expression of any of
the markers disclosed herein is preferably done using HT-FACS.
Antibodies and fluorescently-labelled antibodies for all of the
various markers discussed herein are commercially-available.
[0056] The IMP cells of the invention preferably demonstrate an
antibody mean fluorescence intensity (MFI) of at least 330, such as
at least 350 or at least 400, for MIC A/B, an MFI of at least 210,
such as at least 250 or at least 300, for CD304 (Neuropilin 1), an
MFI of at least 221, such as at least 250 or at least 300, for
CD178 (FAS ligand), an MFI of at least 186, such as at least 200 or
at least 250, for CD289 (Toll-like receptor 9), an MFI of at least
181, such as at least 200 or at least 250, for CD363
(Sphingosine-1-phosphate receptor 1), an MFI of at least 184, such
as at least 200 or at least 250, for CD99, an MFI of at least 300,
such as at least 350 or at least 400, for CD181 (C-X-C chemokine
receptor type 1; CXCR1), an MFI of at least 173, such as at least
200 or at least 250, for epidermal growth factor receptor (EGF-R),
an MFI of at least 236, such as at least 250 or at least 300, for
CXCR2 and an MFI of at least 160, such as at least 200 or at least
250, for CD126. Mean fluorescent intensity (MFI) is a measure of
intensity, time average energy flux measured in watts per square
metre. It is an SI unit. The MFI for each marker is typically
measured using HT-FACS. The MFI for each marker is preferably
measured using HT-FACS as described in the Example.
[0057] In addition to the ten markers specified above, the IMP
cells of the invention typically express detectable levels of one
or more of the other markers shown in Table 1 in the Example. The
IMP cells may express detectable levels of any number and
combination of those markers.
[0058] The IMP cells preferably express detectable levels of one or
more of CD267, CD47, CD51/CD61, CD49f, CD49d, CD146, CD340, Notch2,
CD49b, CD63, CD58, CD44, CD49c, CD105, CD166, HLA-ABC, CD13, CD29,
CD49e, CD73, CD81, CD90, CD98, CD147, CD151 and CD276. The IMP
cells more preferably express detectable levels of one or more of
CD10, CD111, CD267, CD47, CD273, CD51/CD61, CD49f, CD49d, CD146,
CD55, CD340, CD91, Notch2, CD175s, CD82, CD49b, CD95, CD63, CD245,
CD58, CD108, B2-microglobulin, CD155, CD298, CD44, CD49c, CD105,
CD166, CD230, HLA-ABC, CD13, CD29, CD49e, CD59, CD73, CD81, CD90,
CD98, CD147, CD151 and CD276. The IMP cells may express detectable
levels of any number and combination of these markers. The IMP
cells preferably express detectable levels of all of these
markers.
[0059] The IMP cells preferably express detectable levels of one or
more of CD156b, CD61, CD202b, CD130, CD148, CD288, CD337, SSEA-4,
CD349 and CD140b. The IMP cells more preferably express detectable
levels of one or more of CD156b, CD61, CD202b, CD130, CD148, CD288,
CD337, SSEA-4, CD349, CD140b, CD10, CD111, CD267, CD47, CD273,
CD51/CD61, CD49f, CD49d, CD146, CD55, CD340, CD91, Notch2, CD175s,
CD82, CD49b, CD95, CD63, CD245, CD58, CD108, B2-microglobulin,
CD155, CD298, CD44, CD49c, CD105, CD166, CD230, HLA-ABC, CD13,
CD29, CD49e, CD59, CD73, CD81, CD90, CD98, CD147, CD151 and CD276.
The IMP cells may express detectable levels of any number and
combination of these markers. The IMP cells preferably express
detectable levels of all of these markers.
[0060] The IMP cells preferably express detectable levels of one or
more of CD72, CD133, CD192, CD207, CD144, CD41b, FMC7, CD75, CD3e,
CD37, CD158a, CD172b, CD282, CD100, CD94, CD39, CD66b, CD158b,
CD40, CD35, CD15, PAC-1, CLIP, CD48, CD278, CD5, CD103, CD209, CD3,
CD197, HLA-DM, CD20, CD74, CD87, CD129, CDw329, CD57, CD163, TPBG,
CD206, CD243 (BD), CD19, CD8, CD52, CD184, CD107b, CD138, CD7,
CD50, HLA-DR, CD158e2, CD64, DCIR, CD45, CLA, CD38, CD45RB, CD34,
CD101, CD2, CD41a, CD69, CD136, CD62P, TCR alpha beta, CD16b, CD1a,
ITGB7, CD154, CD70, CDw218a, CD137, CD43, CD27, CD62L, CD30, CD36,
CD150, CD66, CD212, CD177, CD142, CD167, CD352, CD42a, CD336,
CD244, CD23, CD45RO, CD229, CD200, CD22, CDH6, CD28, CD18, CD21,
CD335, CD131, CD32, CD157, CD165, CD107a, CD1b, CD332, CD180, CD65
and CD24. The IMP cells may express detectable levels of any number
and combination of these markers. The IMP cells preferably express
detectable levels of all of these markers.
[0061] The IMP cells of the invention are preferably capable of
migrating to a specific, damaged tissue in a patient. In other
words, when the cells are administered to a patient having a
damaged tissue, the cells are capable of migrating (or homing) to
the damaged tissue. This is advantageous because it means that the
cells can be infused via standard routes, for instance
intravenously, and will then target the site of damage. The cells
do not have to be delivered to the damaged tissue. The damage may
be due to injury or disease as discussed in more detail below.
[0062] The specific tissue is preferably cardiac, bone, cartilage,
tendon, ligament, liver, kidney or lung tissue. This applies not
only to migration, but also adherence, transmigration,
proliferation, anti-inflammatory effects and angiogenesis as
discussed in more detail below.
[0063] The ability of the IMP cells of the invention to migrate to
damaged tissue may be measured using standard assays known in the
art. Suitable methods include, but are not limited to, genomic
reverse transcription polymerase chain reaction (RT-PCR with or
without reporter genes) and labelling techniques.
[0064] RT-PCR is the most straightforward and simple means to trace
the IMP cells of the invention within a patient. A transduced
transgene or individual donor markers can be used for this purpose
and transplanted cell-specific signals have been obtained in
several patient studies. The results are generally
semi-quantitative.
[0065] Alternatively, the IMP cells of the invention may be stained
with a dye of interest, such as a fluorescent dye, and may be
monitored in the patient via the signal from the dye. Such methods
are routine in the art.
[0066] Migration (or homing) is typically determined by measuring
the number of cells that arrive at the damaged tissue. It may also
be measured indirectly by observing the numbers of cells that have
accumulated in the lungs (rather than the damaged tissue).
[0067] Damaged heart tissue releases inflammatory chemokines and
cytokines, such as stromal cell-derived factor-1 (SDF-1),
interleukin-8 (IL-8), tumor necrosis factor-alpha (TNF-alpha),
granulocyte-colony-stimulating factor (G-CSF), vascular endothelial
growth factor (VEGF) and hepatocyte growth factor (HGF). In
addition, myocardial infarct increases the levels of VEGF and
erythropoietin (EPO). CXCR4 binds to its ligand SDF-1 and so IMP
cells of the invention expressing CXCR4 will migrate towards the
gradient of SDF-1 generated by the damaged heart tissue. Other
damaged tissues, such as bone, also release SDF-1. If the specific,
damaged tissue is cardiac tissue, the IMP cells of the invention
preferably express detectable levels of CXCR4 or express an
increased amount of CXCR4 compared with MSCs.
[0068] If the specific, damaged tissue is bone tissue, the IMP
cells of the invention preferably express detectable levels of
TGF-beta 3, bone morphogenetic protein-6 (BMP-6), SOX-9,
Collagen-2, CD117 (c-kit), chemokine (C--C motif) ligand 12
(CCL12), CCL7, interleukin-8 (IL-8), platelet-derived growth
factor-A (PDGF-A), PDGF-B, PDGF-C, PDGF-D, macrophage migration
inhibitory factor (MIF), IGF-1, hepatocyte growth factor (HGF),
PDGF-R.alpha., PDGF-R.beta., CXCR4, C--C chemokine receptor type 1
(CCR1), IGF-1 receptor (IGF-1R), hepatocyte growth factor receptor
(HGFR), CXCL12 and NFkappaB. The bone-homing IMP cells of the
invention preferably express an increased amount of one or more of,
or even all of, these factors compared with mesenchymal stem cells
MSCs. The detectable expression of these markers may be measured as
discussed above.
[0069] The IMP cells of the invention are preferably capable of
adhering to a specific, damaged tissue in a patient. Adherence and
adhesion assay are known in the art (Humphries, Methods Mol Biol.
2009; 522:203-10).
[0070] The IMP cells of the invention are preferably capable of
transmigrating through the vascular endothelium to a specific,
damaged tissue in a patient. Transmigration assays are known in the
art (Muller and Luscinskas, Methods Enzymol. 2008; 443:
155-176).
[0071] The IMP cells of the invention are preferably capable of
proliferating in a specific, damaged tissue in a patient. Cell
proliferation assays are well known in the art. Such assays are
commercially available, for instance from Life
Technologies.RTM..
[0072] The IMP cells of the invention are preferably capable of
promoting angiogenesis in a specific, damaged tissue in a patient.
Angiogenesis assays are known in the art (Auerback et al., Clin
Chem. 2003 January; 49(1):32-40).
[0073] The IMP cells of the invention are preferably capable of
having anti-inflammatory effects in a damaged tissue of a patient.
The ability of the IMP cells of the invention to have
anti-inflammatory effects may also be measured using standard
assays known in the art. Suitable methods include, but are not
limited to, enzyme-linked immunosorbent assays (ELISAs) for the
secretion of cytokines, enhanced mixed leukocyte reactions and
up-regulation of co-stimulatory molecules and maturation markers,
measured by flow cytometry. Specific methods that may be used are
disclosed in the Example. The cytokines measured are typically
interleukins, such as interleukin-8 (IL-8), selectins, adhesion
molecules, such as Intercellular Adhesion Molecule-1 (ICAM-1), and
chemoattractant proteins, such as monocyte chemotactic protein-1
(MCP-1) and tumour necrosis factor alpha (TNF-alpha). Assays for
these cytokines are commercially-available. Anti-inflammatory
factors are preferably detected and measured using the Luminex.RTM.
assay described in the Examples. Such assays are commercially
available from Life Technologies.RTM..
[0074] The IMP cells preferably secrete detectable levels of one or
more of interleukin-6 (IL-6), IL-8, C-X-C motif chemokine 10
(CXCL10; interferon gamma-induced protein 10; IP-10), Chemokine
(C--C motif) ligand 2 (CCL2; monocyte chemotactic protein-1; MCP-1)
and Chemokine (C--C motif) ligand 5 (CCL5; regulated on activation,
normal T cell expressed and secreted; RANTES). The IMP cells may
secrete any number and combination of these factors. The IMP cells
preferably secrete all of these markers.
[0075] The IMP cells preferably secrete an increased amount of one
or more of IL-6, IL-8, IP-10, MCP-1 and RANTES compared with a MSC.
The IMP cells may secrete an increased amount of any number and
combination of these factors. The IMP cells preferably secrete an
increased amount of all of these markers.
[0076] The IMP cells preferably secrete a decreased amount of
interleukin-10 (IL-10) and/or IL-12 compared with a mesenchymal
stem cell MSC. IL-10 and IL-12 are pro-inflammatory cytokines.
[0077] The IMP cells of the invention are more preferably capable
of migrating to a damaged tissue in a patient and having
anti-inflammatory effects in the damaged tissue. This allows the
damage to be repaired effectively and reduces the number of cells
that need to be administered.
[0078] The IMP cells of the invention will express a variety of
different other markers over and above those discussed above. Some
of these will assist the IMP cells will their ability to migrate to
a damaged tissue and have anti-inflammatory effects once there. Any
of the IMP cells of the invention may further express detectable
levels of one or more of (i) insulin-like growth factor-1 (IGF-1),
(ii) IGF-1 receptor; (iii) C--C chemokine receptor type 1 (CCR1),
(iv) stromal cell-derived factor-1 (SDF-1), (v) hypoxia-inducible
factor-1 alpha (HIF-1 alpha), (vi) Akt1 and (vii) hepatocyte growth
factor (HGF) and/or granulocyte colony-stimulating factor
(G-CSF).
[0079] IGF-1 receptors promote migration capacity towards an IGF-1
gradient. One of the mechanisms by which IGF-1 increases migration
is by up-regulating CXCR4 on the surface of the cells, which makes
them more sensitive to SDF-1 signaling. This is discussed
above.
[0080] CCR1 is the receptor for CCL7 (previously known as MCP3)
increases homing and engraftment capacity of MSCs (and so would be
expected to have the same effect for the IMP cells of the
invention) and can increase the capillary density in injured
myocardium through paracrine signalling.
[0081] HIF-1 alpha activates pathways that increase oxygen delivery
and promote adaptive pro-survival responses. Among the many target
genes of HIF-1 alpha are erythropoietin (EPO), endothelin and VEGF
(with its receptor Flk-1). IMP cells that express or express an
increased amount of HIF-1 alpha will have upregulated expression of
paracrine stimuli of for example several vasculogenic growth
factors that may promote a more therapeutic subtype. As described
in more detail below, the IMP cells of the invention can be
preconditioned into a more therapeutic subtype by culturing them
under hypoxic conditions (less than 20% oxygen), such as for
example about 2% or about 0% oxygen.
[0082] Akt1 is an intracellular serine/threonine protein kinase
that plays a key role in multiple cellular processes such as
glucose metabolism, cell proliferation, apoptosis, transcription
and cell migration. Overexpression of Akt1 has been shown to
prevent rat MSCs from undergoing apoptosis and will have the same
effect in the IMP cells of the invention. Protection from apoptosis
will enhance the therapeutic effect of the IMP cells.
[0083] The overexpression of HGF by MSCs has been shown to prevent
post-ischemic heart failure by inhibition of apoptosis via
calcineurin-mediated pathway and angiogenesis. HGF and G-CSF
exhibit synergistic effects in this regard. MSCs that have a high
expression of HGF and its receptor c-met also have an increased
migratory capacity into the damaged tissue, achieved through
hormonal, paracrine and autocrine signaling. The same will be true
for the IMP cells of the invention expressing HGF and/or G-CSF.
[0084] The IMP cells may express detectable levels off one or more
of (i) to (vii) defined above. The IMP cells of the invention
preferably express an increased amount of one or more of (i) to
(vii) compared with MSCs. Quantitative assays for cell markers are
described above. The detectable expression of these markers and
their level of expression may be measured as discussed above.
[0085] Any of the IMP cells of the invention may express detectable
levels of one or more of (i) vascular endothelial growth factor
(VEGF), (ii) transforming growth factor beta (TGF-beta), (iii)
insulin-like growth factor-1 (IGF-1), (iv) fibroblast growth factor
(FGF), (v) tumour necrosis factor alpha (TNF-alpha), (vi)
interferon gamma (IFN-gamma) and (vii) interleukin-1 alpha (IL-1
alpha). Conditioned medium from cells overexpressing VEGF has been
shown to alleviate heart failure in a hamster model. Hence, the IMP
cells of the invention which express or express an increased amount
of VEGF will have the same effect of damaged cardiac tissue.
[0086] The IMP cells may express detectable levels of one or more
of (i) to (vii). The IMP cells of the invention may express an
increased amount of one or more of (i) to (vii) compared with MSCs.
Quantitative assays for cell markers are described above. The
detectable expression of these markers and their level of
expression may be measured as discussed above.
[0087] In both sets of definitions of (i) to (vii) given above, any
combination of one or more of (i) to (vii) may be expressed or
expressed in an increased amount. For instance, for each definition
of (i) to (vii), the IMP cells may express detectable levels of, or
express an increased amount of, (i); (ii); (iii); (iv); (v); (vi);
(vii); (i) and (ii); (i) and (iii); (i) and (iv); (i) and (v); (i)
and (vi); (i) and (vii); (ii) and (iii); (ii) and (iv); (ii) and
(v); (ii) and (vi); (ii) and (vii); (iii) and (iv); (iii) and (v);
(iii) and (vi); (iii) and (vii); (iv) and (v); (iv) and (vi); (iv)
and (vii); (v) and (vi); (v) and (vii); (vi) and (vii); (i), (ii)
and (iii); (i), (ii) and (iv); (i), (ii) and (v); (i), (ii) and
(vi); (i), (ii) and (vii); (i,), (iii) and (iv); (i), (iii) and
(v); (i), (iii) and (vi); (i), (iii) and (vii); (i), (iv) and (v);
(i), (iv) and (vi); (i), (iv) and (vii); (i), (v) and (vi); (i),
(v) and (vii); (i), (vi) and (vii); (ii), (iii) and (iv); (ii),
(iii) and (v); (ii), (iii) and (vi); (ii), (iii) and (vii); (ii),
(iv) and (v); (ii), (iv) and (vi); (ii), (iv) and (vii); (ii), (v)
and (vi); (ii), (v) and (vii); (ii), (vi) and (vii); (iii), (iv)
and (v); (iii), (iv) and (vi); (iii), (iv) and (vii); (iii), (v)
and (vi); (iii), (v) and (vii); (iii), (vi) and (vii); (iv), (v)
and (vi); (iv), (v) and (vii); (iv), (vi) and (vii); (v), (vi) and
(vii); (i), (ii), (iii) and (iv); (i), (ii), (iii) and (v); (i),
(ii), (iii) and (vi); (i), (ii), (iii) and (vii); (i), (ii), (iv)
and (v); (i), (ii), (iv) and (vi); (i), (ii), (iv) and (vii); (i),
(ii), (v) and (vi); (i), (ii), (v) and (vii); (i), (ii), (vi) and
(vii); (i), (iii), (iv) and (v); (i), (iii), (iv) and (vi); (i),
(iii), (iv) and (vii); (i), (iii), (v) and (vi); (i), (iii), (v)
and (vii); (i), (iii), (vi) and (vii); (i), (iv), (v) and (vi);
(i), (iv), (v) and (vii); (i), (iv), (vi) and (vii); (i), (v), (vi)
and (vii); (ii), (iii), (iv) and (v); (ii), (iii), (iv) and (vi);
(ii), (iii), (iv) and (vii); (ii), (iii), (v) and (vi); (ii),
(iii), (v) and (vii); (ii), (iii), (vi) and (vii); (ii), (iv), (v)
and (vi); (ii), (iv), (v) and (vii); (ii), (iv), (vi) and (vii);
(ii), (v), (vi) and (vii); (iii), (iv), (v) and (vi); (iii), (iv),
(v) and (vii); (iii), (iv), (vi) and (vii); (iii), (v), (vi) and
(vii); (iv), (v), (vi) and (vii); (i), (ii), (iii), (iv) and (v);
(i), (ii), (iii), (iv) and (vi); (i), (ii), (iii), (iv) and (vii);
(i), (ii), (iii), (v) and (vi); (i), (ii), (iii), (v) and (vii);
(i), (ii), (iii), (vi) and (vii); (i), (ii), (iv), (v) and (vi);
(i), (ii), (iv), (v) and (vii); (i), (ii), (iv), (vi) and (vii);
(i), (ii), (v), (vi) and (vii); (i), (iii), (iv), (v) and (vi);
(i), (iii), (iv), (v) and (vii); (i), (iii), (iv), (vi) and vii);
(i), (iii), (v), (vi) and (vii); (i), (iv), (v), (vi) and (vii);
(ii), (iii), (iv), (v) and (vi); (ii), iii), (iv), (v) and (vii);
(ii), (iii), (iv), (vi) and (vii); (ii), (iii), (v), (vi) and
(vii); (ii), (iv), (v), (vi) and (vii); (iii), (iv), (v), (vi) and
vii); (i), (ii), (iii), (iv), (v) and (vi); (i), (ii), (iii), (iv),
(v) and (vii); (i), (ii), (iii), (iv), (vi) and (vii); (i), (ii),
(iii), (v), (vi) and (vii); (i), (ii), (iv), (v), (vi) and (vii);
(i), (iii), (iv), (v), (vi) and (vii); (ii), (iii), (iv), (v), (vi)
and (vii); or (i), (ii), (iii), (iv), (v), (vi) and (vii). The
combinations for each definition of (i) to (vii) are independently
selectable from this list.
[0088] In addition to any of the markers discussed above, the IMP
cells of the invention preferably also express detectable levels
of, LIF and/or platelet-derived growth factor (PDGF) receptors. The
IMP cells of the invention preferably express an increased amount
of LIF and/or platelet-derived growth factor (PDGF) receptors
compared with mesenchymal stem cells. The PDGF receptors are
preferably PDGF-A receptors and/or PSDGF-B receptors. MSCs that
have high expression of these receptors can migrate effectively
into areas in which platelets have been activated, such as wounds
and thrombotic vessels. The same will be true of IMP cells
expressing or expressing an increased amount of the receptors.
[0089] The IMP cells of the invention are preferably autologous. In
other words, the cells are preferably derived from the patient into
which the cells will be administered. Alternatively, the IMP cells
are preferably allogeneic. In other words, the cells are preferably
derived from a patient that is immunologically compatible with the
patient into which the cells will be administered. An IMP cell of
the invention may be isolated, substantially isolated, purified or
substantially purified. The IMP cell is isolated or purified if it
is completely free of any other components, such as culture medium,
other cells of the invention or other cell types. The IMP cell is
substantially isolated if it is mixed with carriers or diluents,
such as culture medium, which will not interfere with its intended
use. Alternatively, the IMP cell of the invention may be present in
a growth matrix or immobilized on a surface as discussed below.
[0090] IMP cells of the invention may be isolated using a variety
of techniques including antibody-based techniques. Cells may be
isolated using negative and positive selection techniques based on
the binding of monoclonal antibodies to those surface markers which
are present on the IMP cell (see above). Hence, the IMP cells may
be separated using any antibody-based technique, including
fluorescent activated cell sorting (FACS) and magnetic bead
separation.
[0091] As discussed in more detail below, the IMP cells may be
treated ex vivo. Thus the cells may be loaded or transfected with a
therapeutic or diagnostic agent and then used therapeutically in
the methods of the invention.
Population of the Invention
[0092] The invention also provides a population of two or more IMP
cells of the invention. Any number of cells may be present in the
population. The population of the invention preferably comprises at
least about 5.times.10.sup.5 IMP cells of the invention. The
population more preferably comprises at least about
1.times.10.sup.6, at least about 2.times.10.sup.6, at least about
2.5 2.times.10.sup.6, at least about 5.times.10.sup.6, at least
about 1.times.10.sup.7, at least about 2.times.10.sup.7, at least
about 5.times.10.sup.7, at least about 1.times.10.sup.8 or at least
about 2.times.10.sup.8 IMP cells of the invention. In some
instances, the population may comprise at least about
1.0.times.10.sup.7, at least about 1.0.times.10.sup.8, at least
about 1.0.times.10.sup.9, at least about 1.0.times.10.sup.10, at
least about 1.0.times.10.sup.11 or at about least
1.0.times.10.sup.12 IMP cells of the invention or even more.
[0093] The population comprising two or more IMP cells of the
invention may comprise other cells in addition to the IMP cells of
the invention. However, at least 70% of the cells in the population
are preferably IMP cells of the invention. More preferably, at
least about 75%, at least about 80%, at least about 85%, at least
about 90%, at least about 97%, at least about 98% or at least about
99% of the cells in the population are IMP cells of the
invention.
[0094] The invention also provides specific populations of IMP
cells. The invention provides a population of immuno-modulatory
progenitor (IMP) cells, wherein [0095] (i) at least 90%, preferably
at least 97% and more preferably at least 97.1%, of the cells in
the population express detectable levels of MIC A/B, [0096] (ii) at
least 60%, preferably at least 65% and more preferably at least
65.2%, of the cells in the population express detectable levels of
CD304 (Neuropilin 1), [0097] (iii) at least 45%, preferably at
least 51% and more preferably at least 51.6%, of the cells in the
population express detectable levels of CD178 (FAS ligand), [0098]
(iv) at least 10%, preferably at least 11% and more preferably at
least 11.3%, of the cells in the population express detectable
levels of CD289 (Toll-like receptor 9), [0099] (v) at least 15%,
preferably at least 18% and more preferably at least 18.7%, of the
population express detectable levels of CD363
(Sphingosine-1-phosphate receptor 1), [0100] (vi) at least 20%,
preferably at least 24% and more preferably at least 24.8%, of the
cells in the population express detectable levels of CD99, [0101]
(vii) at least 80%, preferably at least 85%, of the cells in the
population express detectable levels of CD181 (C-X-C chemokine
receptor type 1; CXCR1), [0102] (viii) at least 30%, preferably at
least 33% and more preferably at least 33.3%, of the cells in the
population express detectable levels of epidermal growth factor
receptor (EGF-R), [0103] (xi) at least 60%, preferably at least 68%
and more preferably at least 68.8%, of the cells in the population
express detectable levels of CXCR2 and [0104] (x) at least 5%,
preferably at least 7% and more preferably at least 7.05%, of the
cells in the population express detectable levels of CD126.
[0105] The cells in these preferred populations may further express
detectable levels of any of the markers discussed above with
reference to the IMP of the invention. The cells in the these
preferred populations may have any of the advantageous properties
of the IMP cells discussed above.
[0106] At least 90%, such as at least 95%, of the cells in the
population preferably express detectable levels of one or more of
CD10, CD111, CD267, CD47, CD273, CD51/CD61, CD49f, CD49d, CD146,
CD55, CD340, CD91, Notch2, CD175s, CD82, CD49b, CD95, CD63, CD245,
CD58, CD108, B2-microglobulin, CD155, CD298, CD44, CD49c, CD105,
CD166, CD230, HLA-ABC, CD13, CD29, CD49e, CD59, CD73, CD81, CD90,
CD98, CD147, CD151 and CD276. At least 90%, such as at least 95%,
of the cells in the population may express detectable levels of any
number and combination of these markers. At least 90%, such as at
least 95%, of the cells in the population preferably express
detectable levels of all of these markers.
[0107] At least 80%, such as at least 85%, of the cells in the
population preferably express detectable levels of one or more of
CD156b, CD61, CD202b, CD130, CD148, CD288, CD337, SSEA-4, CD349 and
CD140b. At least 80%, such as at least 85%, of the cells in the
population may express detectable levels of any number and
combination of these markers. At least 80%, such as at least 85%,
of the cells in the population preferably express detectable levels
of all of these markers.
[0108] At least 70%, such as at least 75%, of the cells in the
population preferably express detectable levels of one or more of
CD318, CD351, CD286, CD46, CD119 and CD132. At least 70%, such as
at least 75%, of the cells in the population may express detectable
levels of any number and combination of these markers. At least
70%, such as at least 75%, of the cells in the population
preferably express detectable levels of all of these markers.
[0109] 1% or fewer, such as 0.5% or fewer, of the cells in the
population preferably express detectable levels of one or more of
CD72, CD133, CD192, CD207, CD144, CD41b, FMC7, CD75, CD3e, CD37,
CD158a, CD172b, CD282, CD100, CD94, CD39, CD66b, CD158b, CD40,
CD35, CD15, PAC-1, CLIP, CD48, CD278, CD5, CD103, CD209, CD3,
CD197, HLA-DM, CD20, CD74, CD87, CD129, CDw329, CD57, CD163, TPBG,
CD206, CD243 (BD), CD19, CD8, CD52, CD184, CD107b, CD138, CD7,
CD50, HLA-DR, CD158e2, CD64, DCIR, CD45, CLA, CD38, CD45RB, CD34,
CD101, CD2, CD41a, CD69, CD136, CD62P, TCR alpha beta, CD16b, CD1a,
ITGB7, CD154, CD70, CDw218a, CD137, CD43, CD27, CD62L, CD30, CD36,
CD150, CD66, CD212, CD177, CD142, CD167, CD352, CD42a, CD336,
CD244, CD23, CD45RO, CD229, CD200, CD22, CDH6, CD28, CD18, CD21,
CD335, CD131, CD32, CD157, CD165, CD107a, CD1b, CD332, CD180, CD65
and CD24. 1% or fewer, such as 0.5% or fewer, of the cells in the
population may express detectable levels of any number and
combination of these markers. 1% or fewer, such as 0.5% or fewer,
of the cells in the population preferably express detectable levels
of all of these markers.
[0110] In any of the embodiments above where populations are
defined with reference to % of cells expressing certain markers,
the populations preferably comprise at least 5,000 cells, such as
at least 6,000, at least 7,000, at least 8,000, at least 9,000, at
least 10,000, at least 20,000, at least 30,000 or at least 40,000
cells. These populations may comprise any of the number of cells
discussed above.
[0111] Any of the populations of cells disclosed herein may be
diluted with other cells before use. For instance, the population
may be combined with patient blood, mononuclear cells (MCs), MSCs,
progenitor cells of the mesodermal lineage (PMLs) or a combination
thereof. PMLs are disclosed in PCT/GB2012/051600 (published as WO
2013/005053).
[0112] The populations of the invention are advantageous for
therapy as discussed below. This ability to produce populations
comprising large numbers of IMP cells of the invention is one of
the key advantages of the invention. The invention allows the
treatment of patients with a population of cells of which most, if
not all, migrate efficiently to the tissue of interest and have
anti-inflammatory effects once there. This allows the use of a low
cell-dose and avoids off-target side effects and volume-related
side effects.
[0113] The population of the invention is preferably homologous. In
other words, all of the IMP cells in the population are preferably
genotypically and phenotypically identical. The population is
preferably autologous or allogeneic as defined above.
[0114] However, the population can also be semi-allogeneic.
Semi-allogeneic populations are typically produced from mononuclear
cells from two or more patients that are immunologically compatible
with the patient into which the population will be administered. In
other words, all of the cells in the population are preferably
genetically identical or sufficiently genetically identical that
the population is immunologically compatible with the patient into
which the population will be administered. Since the IMP cells of
the invention may be derived from a patient, they may be autologous
with the patient to be treated (i.e. genetically identical with the
patient or sufficiently genetically identical that they are
compatible for administration to the patient).
[0115] The population of the invention may be isolated,
substantially isolated, purified or substantially purified. A
population is isolated or purified if it is completely free of any
other components, such as culture medium and other cells. A
population is substantially isolated if it is mixed with carriers
or diluents, such as culture medium, which will not interfere with
its intended use. Other carriers and diluents are discussed in more
detail below. A substantially isolated or substantially purified
population does not comprise cells other than the IMP cells of the
invention. In some embodiments, the population of the invention may
be present in a growth matrix or immobilized on a surface as
discussed below.
[0116] The population is typically cultured in vitro. Techniques
for culturing cells are well known to a person skilled in the art.
The cells are may be cultured under standard conditions of
37.degree. C., 5% CO.sub.2 in medium without serum. The cells are
preferably cultured under low oxygen conditions as discussed in
more detail below. The cells may be cultured in any suitable flask
or vessel, including wells of a flat plate such as a standard 6
well plate. Such plates are commercially available from Fisher
scientific, VWR suppliers, Nunc, Starstedt or Falcon. The wells
typically have a capacity of from about 1 ml to about 4 ml.
[0117] The flask, vessel or wells within which the population is
contained or cultured may be modified to facilitate handling of the
IMP cells. For instance, the flask, vessel or wells may be modified
to facilitate culture of the cells, for instance by including a
growth matrix. The flask, vessel or wells may be modified to allow
attachment of the IMP cells or to allow immobilization of the IMP
cells onto a surface. One or more surfaces may be coated with
extracellular matrix proteins such as laminin or collagen or any
other capture molecules that bind to the cells and immobilize or
capture them on the surface(s).
[0118] The population may be modified ex vivo using any of the
techniques described herein. For instance, the population may be
transfected or loaded with therapeutic or diagnostics agents. The
population may then be used in the methods of treatment discussed
in more detail below.
Method of Producing an IMP Cell of the Invention
[0119] The invention also provides a method for producing a
population of the invention. The method involves culturing
mononuclear cells (MCs) under conditions which induce the MCs to
differentiate into IMP cells. The method then involves harvesting
and culturing the IMP cells which expresses detectable levels of
MIC A/B, CD304 (Neuropilin 1), CD178 (FAS ligand), CD289 (Toll-like
receptor 9), CD363 (Sphingosine-1-phosphate receptor 1), CD99,
CD181 (C-X-C chemokine receptor type 1; CXCR1), epidermal growth
factor receptor (EGF-R), CXCR2 and CD126. The harvested cells may
express detectable levels of or increased amounts of any of the
markers and factors described above with reference to the cells of
the invention.
[0120] Mononuclear cells (MCs) and methods of isolating them are
known in the art. The MCs may be primary MCs isolated from bone
marrow. The MCs are preferably peripheral blood MCs (PBMCs), such
as lymphocytes, monocytes and/or macrophages. PBMCs can be isolated
from blood using a hydrophilic polysaccharide, such as Ficoll.RTM..
For instance, PBMCs may be isolated from blood using
Ficoll-Paque.RTM. (a commercially-available density medium) as
disclosed in the Example.
[0121] Before they are cultured, the MCs may be exposed to a
mesenchymal stem cell enrichment cocktail. The cocktail preferably
comprises antibodies that recognise CD3, CD14, CD19, CD38, CD66b
(which are present on unwanted cells) and a component of red blood
cells. Such a cocktail cross links unwanted cells with red blood
cells forming immunorosettes which may be removed from the wanted
MCs. A preferred cocktail is RosetteSep.RTM..
[0122] Conditions suitable for inducing MCs to differentiate into
mesenchymal cells (tissue mainly derived from the mesoderm) are
known in the art. For instance, suitable conditions are disclosed
in Capelli, C., et al. (Human platelet lysate allows expansion and
clinical grade production of mesenchymal stromal cells from small
samples of bone marrow aspirates or marrow filter washouts. Bone
Marrow Transplantation, 2007. 40: p. 785-791). These conditions may
also be used to induce MCs to differentiate into IMP cells in
accordance with the invention.
[0123] The method preferably comprises culturing MCs with plasma
lysate to induce the MCs to differentiate into IMP cells. Platelet
lysate refers to the combination of natural growth factors
contained in platelets that has been released through lysing those
platelets. Lysis can be accomplished through chemical means (i.e.
CaCl.sub.2), osmotic means (use of distilled H.sub.2O) or through
freezing/thawing procedures. Platelet lysate can be derived from
whole blood as described in U.S. Pat. No. 5,198,357. Platelet
lysate is preferably prepared as described in PCT/GB12/052911
(published as WO 2013/076507). The plasma lysate is preferably
human plasma lysate.
[0124] In a preferred embodiment, step (a) of the method of the
invention comprises culturing MCs in a medium comprising platelet
lysate for sufficient time to induce the MCs to differentiate into
IMP cells. The sufficient time is typically from about 15 to about
25 days, preferably about 22 days. The medium preferably comprises
about 20% or less platelet lysate by volume, such as about 15% or
less by volume or about 10% or less by volume. The medium
preferably comprises from about 5% to about 20% of platelet lysate
by volume, such as from about 10% to about 15% by volume. The
medium preferably comprises about 10% of platelet lysate by
volume.
[0125] In another preferred embodiment, step (a) of the method of
the invention comprises exposing MCs to a mesenchymal enrichment
cocktail and then culturing the MCs in a medium comprising platelet
lysate for sufficient time to induce the MCs to differentiate into
IMP cells. The sufficient time is typically from about 15 to about
25 days, preferably about 22 days.
[0126] In step (a), the medium is preferably Minimum Essential
Medium (MEM). MEM is commercially available from various sources
including Sigma-Aldrich. The medium preferably further comprises
one or more of heparin, L-glutamine and penicillin/streptavidin
(P/S). The L-glutamine may be replaced with GlutaMAX.RTM. (which is
commercially-available from Life Technologies).
[0127] As discussed above, some of the IMP cells of the invention
express detectable levels of CXCR4. Expression of CXCR4 is
cytokine-dependent and is increased when cells are exposed to stem
cell factor (SCF), interleukin-6 (IL-6), Flt-3 ligand, hepatocyte
growth factor (HGF) and IL-3. The medium may comprise one or more
of (i) SCF, (ii) IL-6, (iii) Flt-3 ligand, (iv) hepatocyte growth
factor and (v) IL-3, such as (i); (ii); (iii); (iv); (v); (i) and
(ii); (i) and (iii); (i) and (iv); (i) and (v); (ii) and (iii);
(ii) and (iv); (ii) and (v); (iii) and (iv); (iii) and (v); (iv)
and (v); (i), (ii) and (iii); (i), (ii) and (iv); (i), (ii) and
(v); (i), (iii) and (iv); (i), (iii) and (v); (i), (iv) and (v);
(ii), (iii) and (iv); (ii), (iii) and (v); (ii), (iv) and (v);
(iii), (iv) and (v); or (i), (ii), (iii), (iv) and (v). Any of (i)
to (v) may be present at from about from about 10 to about about
150 ng/ml.
[0128] Step (a) preferably comprises culturing the MCs under
conditions which allow the IMP cells to adhere. Suitable conditions
are discussed in more detail above.
[0129] In step (a), the MCs are preferably cultured under low
oxygen conditions. The MCs are preferably cultured at less than
about 20% oxygen (O.sub.2), such as less than about 19%, less than
about 18%, less than about 17%, less than about 16%, less than
about 15%, less than about 14%, less than about 13%, less than
about 12%, less than about 11%, less than about 10%, less than
about 9%, less than about 8%, less than about 7%, less than about
6%, less than about 5%, less than about 4%, less than about 3%,
less than about 2% or less than about 1% oxygen (O.sub.2). The MCs
are preferably cultured at from about 0% to about 19% O.sub.2, such
as from about 1% to about 15% O.sub.2, from about 2% to about 10%
02 or from about 5% to about 8% O.sub.2. The MCs are most
preferably cultured at about 0% O.sub.2. The figures for % oxygen
(or % O.sub.2) quoted above relate to % by volume of oxygen in the
gas supplied to the cells during culture, for instance by the cell
incubator. It is possible that some oxygen may leak into the
incubator or enter when the door is opened.
[0130] In step (a), the MCs are most preferably cultured in the
presence of platelet lysate and under low oxygen conditions. This
combination mimics the natural conditions in the damaged tissue and
so result in healthier and more therapeutically potent cells.
Conventional cell culture is performed in 20% or 21% oxygen
(approximately the atmospheric content) but there is no place in
the human body that has this oxygen level. The epithelial cells in
the lungs would "see" this oxygen level, but once the oxygen is
dissolved and leaves the lungs, it decreases to around 17%. From
there, it decreases even further to about 1-2% in the majority of
the tissues, but being as low as 0.1% in avascular tissues such as
the cartilage in the joints.
[0131] In step (b), the method further comprises harvesting and
culturing IMP cells which have the necessary marker expression
pattern as discussed above. The IMP cells having the necessary
marker expression pattern may be harvested using any antibody-based
technique, including fluorescent activated cell sorting (FACS) and
magnetic bead separation. FACS is preferred. HT-FACS is more
preferred.
[0132] Any of the methods for culturing IMP cells disclosed in
relation to step (a) equally apply to step (b). In particular, the
cells are cultured in step (b) in the presence of platelet lysate
and under low oxygen conditions as discussed above in relation to
step (a).
[0133] As will be clear from the discussion above, the method of
the invention is carried out in clinically relevant conditions,
i.e. in the absence of trace amounts of endotoxins and other
environmental contaminants, such as lipopolysaccharides,
lipopeptides and peptidoglycans, etc. This makes the IMP cells of
the invention particularly suitable for administration to
patients.
[0134] The MCs are preferably obtained from a patient or an
allogeneic donor. The invention also provides a method for
producing a population of the invention that is suitable for
administration to a patient, wherein the method comprises culturing
MCs obtained from the patient under conditions which induce the MCs
to differentiate into IMP cells and (b) harvesting and culturing
those progenitor cells which have an expression pattern as defined
above and thereby producing a population of the invention that is
suitable for administration to the patient. The population will be
autologous with the patient and therefore will not be rejected upon
implantation. The invention also provides a population of the
invention that is suitable for administration to a patient and is
produced in this manner.
[0135] Alternatively, the invention provides a method for producing
a population of the invention that is suitable for administration
to a patient, wherein the method comprises culturing MCs obtained
from a different patient that is immunologically compatible with
the patient into which the cells will be administered under
conditions which induce the MCs to differentiate into IMP cells and
(b) harvesting and culturing those IMP cells which have an
expression pattern as defined above and thereby producing a
population of the invention that is suitable for administration to
the patient. The population will be allogeneic with the patient and
therefore will reduce the chance of rejection upon implantation.
The invention also provides a population of the invention that is
suitable for administration to a patient and is produced in this
manner.
Medicaments, Methods and Therapeutic Use
[0136] The IMP cells of the invention may be used in a method of
therapy of the human or animal body. Thus the invention provides an
IMP cell of the invention or a population of the invention for use
in a method of treatment of the human or animal body by therapy. In
particular, the invention concerns using the IMP cells of the
invention or a population of the invention to repair a damaged
tissue in a patient. The invention also concerns using the IMP
cells of the invention or a population of the invention to treat a
cardiac, bone, cartilage, tendon, ligament, liver, kidney or lung
injury or disease in the patient.
[0137] The invention provides a method of repairing a damaged
tissue in a patient, comprising administering to the patient a
population of the invention, wherein the population comprises a
therapeutically effective number of cells, and thereby treating the
damaged tissue in the patient. The invention also provides a
population of the invention for use in repairing a damaged tissue
in the patient. The invention also provides use of a population of
the invention in the manufacture of a medicament for repairing a
damaged tissue in a patient.
[0138] The tissue is preferably derived from the mesoderm. The
tissue is more preferably cardiac, bone, cartilage, tendon,
ligament, liver, kidney or lung tissue.
[0139] The damage to the tissue may be caused by injury or disease.
The injury or disease is preferably a cardiac, bone, cartilage,
tendon, ligament, liver, kidney or lung injury or disease in a
patient. The invention therefore provides a method of treating a
cardiac, bone, cartilage, tendon, ligament, liver, kidney or lung
injury or disease in a patient, comprising administering to the
patient a population of the invention, wherein the population
comprises a therapeutically effective number of cells, and thereby
treating the injury or disease in the patient. The invention also
provides a population of the invention for use in treating a
cardiac, bone, cartilage, tendon, ligament, liver, kidney or lung
injury or disease in a patient. The invention also provides use of
a population of the invention in the manufacture of a medicament
for treating a cardiac, bone, cartilage, tendon, ligament, liver,
kidney or lung injury or disease in a patient.
[0140] The cardiac injury or disease is preferably selected from
myocardial infarct (MI), left ventricular hypertrophy, right
ventricular hypertrophy, emboli, heart failure, congenital heart
deficit, heart valve disease, arrhythmia and myocarditis.
[0141] MI increases the levels of VEGF and EPO released by the
myocardium. Furthermore, MI is associated with an inflammatory
reaction and infarcted tissue also releases macrophage migration
inhibitory factor (MIF), interleukin (IL-6) and KC/Gro-alpha. CCL7
(previously known as MCP3), CXCL1, CXCL2 are significantly
upregulated in the heart following myocardial infarct (MI) and
might be implicated in regulating engraftment and homing of MSCs to
infarcted myocardium.
[0142] In a myocardial infarct mice model, IL-8 was shown to highly
up-regulate gene expression primarily in the first 2 days post-MI.
Remarkably, the increased IL-8 expression was located predominantly
in the infarcted area and the border zone, and only to a far lesser
degree in the spared myocardium. By activating CXCR2, MIF displays
chemokine-like functions and acts as a major regulator of
inflammatory cell recruitment and atherogenesis.
[0143] The bone disease or injury is preferably selected from
fracture, Salter-Harris fracture, greenstick fracture, bone spur,
craniosynostosis, Coffin-Lowry syndrome, fibrodysplasia ossificans
progressive, fibrous dysplasia, Fong Disease (or Nail-patella
syndrome), hypophosphatasia, Klippel-Feil syndrome, Metabolic Bone
Disease, Nail-patella syndrome, osteoarthritis, osteitis deformans
(or Paget's disease of bone), osteitis fibrosa cystica (or Osteitis
fibrosa or Von Recklinghausen's disease of bone), osteitis pubis,
condensing osteitis (or osteitis condensans), osteitis condensans
ilii, osteochondritis dissecans, osteogenesis imperfecta,
osteomalacia, osteomyelitis, osteopenia, osteopetrosis,
osteoporosis, osteonecrosis, porotic hyperostosis, primary
hyperparathyroidism, renal osteodystrophy, bone cancer, a bone
lesion associated with metastatic cancer, Gorham Stout disease,
primary hyperparathyroidism, periodontal disease, and aseptic
loosening of joint replacements. The bone cancer can be Ewing
sarcoma, multiple myeloma, osteosarcoma (giant tumour of the bone),
osteochondroma or osteoclastoma. The metastatic cancer that results
in a bone lesion can be breast cancer, prostate cancer, kidney
cancer, lung cancer and/or adult T-cell leukemia.
[0144] If the damaged tissue is cardiac tissue or bone tissue, the
IMP cells in the population preferably express detectable levels of
CD29, CD44, CD73, CD90, CD105, CD271, CXCR1, CXCR2 and CXCR4 and do
not express detectable levels of CD14, CD34 and CD45. If the
damaged tissue is bone tissue, the IMP cells in the population more
preferably express detectable levels of CD29, CD44, CD73, CD90,
CD105, CD271, TGF-beta 3, bone morphogenetic protein-6 (BMP-6),
SOX-9, Collagen-2, CD117 (c-kit), chemokine (C--C motif) ligand 12
(CCL12), CCL7, interleukin-8 (IL-8), platelet-derived growth
factor-A (PDGF-A), PDGF-B, PDGF-C, PDGF-D, macrophage migration
inhibitory factor (MIF), IGF-1, hepatocyte growth factor (HGF),
PDGF-R.alpha., PDGF-R.beta., CXCR4, C--C chemokine receptor type 1
(CCR1), IGF-1 receptor (IGF-1R), hepatocyte growth factor receptor
(HGFR), CXCL12 and NFkappaB and do not express detectable levels of
CD14, CD34 and CD45.
[0145] The disease or disorder may be periodontal disease,
endometriosis or meniscal tears.
[0146] In all instances, the IMP cells of the invention are
preferably derived from the patient or an allogeneic donor.
Deriving the IMP cells of the invention from the patient should
ensure that the IMP cells are themselves not rejected by the
patient's immune system. Any difference between the donor and
recipient will ultimately cause clearance of the IMP cells, but not
before they have repaired at least a part of the damaged
tissue.
[0147] The invention concerns administering to the patient a
therapeutically effective number of IMP cells of the invention to
the patient. A therapeutically effective number is a number which
ameliorates one or more symptoms of the damage, disease or injury.
A therapeutically effective number is preferably a number which
repairs the damaged tissue or treats the disease or injury.
Suitable numbers are discussed in more detail below.
[0148] The IMP cells of the invention may be administered to any
suitable patient. The patient is generally a human patient. The
patient may be any of the animals or mammals mentioned above with
reference to the source of the IMP cells.
[0149] The patient may be an infant, a juvenile or an adult. The
patient may be known to have a damaged tissue or is suspected of
having a damaged tissue. The patient may be susceptible to, or at
risk from, the relevant disease or injury. For instance, the
patient may be genetically predisposed to heart failure.
[0150] The invention may be used in combination with other means
of, and substances for, repairing damaged tissue or providing pain
relief. In some cases, the IMP cells of the invention may be
administered simultaneously, sequentially or separately with other
substances which are intended for repairing the damaged tissue or
for providing pain relief. The IMP cells may be used in combination
with existing treatments for damaged tissue and may, for example,
be simply mixed with such treatments. Thus the invention may be
used to increase the efficacy of existing treatments of damaged
tissue.
[0151] The invention preferably concerns the use of IMP cells
loaded or transfected with a therapeutic and/or diagnostic agent. A
therapeutic agent may help to repair the damaged tissue. A
diagnostic agent, such as a fluorescent molecule, may help to
identify the location of the IMP cells in the patient. The IMP
cells may be loaded or transfected using any method known in the
art. The loading of IMP cells may be performed in vitro or ex vivo.
In each case, the IMP cells may simply be in contact with the agent
in culture. Alternatively, the IMP cells may be loaded with an
agent using delivery vehicle, such as liposomes. Such vehicles are
known in the art.
[0152] The transfection of IMP cells may be performed in vitro or
ex vivo. Alternatively, stable transfection may be performed at the
MC stage allowing IMP cells expressing the transgene to be
differentiated from them. The IMP cells are transfected with a
nucleic acid encoding the agent. For instance, viral particles or
other vectors encoding the agent may be employed. Methods for doing
this are known in the art.
[0153] The nucleic acid gives rise to expression of the agent in
the IMP cells. The nucleic acid molecule will preferably comprise a
promoter which is operably linked to the sequences encoding the
agent and which is active in the IMP cells or which can be induced
in the IMP cells.
[0154] In a particularly preferred embodiment, the nucleic acid
encoding the agent may be delivered via a viral particle. The viral
particle may comprise a targeting molecule to ensure efficient
transfection. The targeting molecule will typically be provided
wholly or partly on the surface of the virus in order for the
molecule to be able to target the virus to the IMP cells.
[0155] Any suitable virus may be used in such embodiments. The
virus may, for example, be a retrovirus, a lentivirus, an
adenovirus, an adeno-associated virus, a vaccinia virus or a herpes
simplex virus. In a particularly preferred embodiment the virus may
be a lentivirus. The lentivirus may be a modified HIV virus
suitable for use in delivering genes. The lentivirus may be a SIV,
FIV, or equine infectious anemia virus (EQIA) based vector. The
virus may be a moloney murine leukaemia virus (MMLV). The viruses
used in the invention are preferably replication deficient.
[0156] Viral particles do not have to be used. Any vector capable
of transfecting the IMP cells of the invention may be used, such as
conventional plasmid DNA or RNA transfection.
[0157] Uptake of nucleic acid constructs may be enhanced by several
known transfection techniques, for example those including the use
of transfection agents. Examples of these agents includes cationic
agents, for example, calcium phosphate and DEAE-Dextran and
lipofectants, for example, lipofectAmine, fugene and
transfectam.
[0158] The cell may be loaded or tranfected under suitable
conditions. The cell and agent or vector may, for example, be
contacted for between five minutes and ten days, preferably from an
hour to five days, more preferably from five hours to two days and
even more preferably from twelve hours to one day.
[0159] The invention also provides IMP cells which have been loaded
or transfected with an agent as discussed above. Such IMP cells may
be used in the therapeutic embodiments of the invention.
[0160] In some embodiments, MCs may be recovered from a patient,
converted into IMP cells using the invention, loaded or transfected
in vitro and then returned to the same patient. In such instances,
the IMP cells employed in the invention, will be autologous cells
and fully matched with the patient. In a preferred case, the cells
employed in the invention are recovered from a patient and utilised
ex vivo and subsequently returned to the same patient.
Pharmaceutical Compositions and Administration
[0161] The invention additionally provides a pharmaceutical
composition comprising an IMP cell of the invention or a population
of the invention in combination with a pharmaceutically acceptable
carrier or diluent, (ii) one or more lipsomes and/or (iii) one or
more microbubbles. The composition may comprise (i); (ii); (iii);
(i) and (ii); (i) and (iii); (ii) and (iii); or (i), (ii) and
(iii). The IMP cell or population are preferably contained with the
one or more liposomes and/or one or more microbubbles. Any number
of liposomes and/or microbubbles may be present. Any of the numbers
discussed above with reference to the population of the invention
are equally application to the lipsomes and/or microbubbles. A
lipsome or microbubble may contain one IMP cell or more than one
IMP cell.
[0162] The composition may comprise any of the IMP cells or
populations mentioned herein and, in some embodiments, the nucleic
acid molecules, vectors, or viruses described herein. The invention
provides a method of repairing a damaged tissue in a patient
comprising administering to the patient an effective amount of a
pharmaceutical composition of the invention. Any of the therapeutic
embodiments discussed above equally apply to this embodiment.
[0163] The various compositions of the invention may be formulated
using any suitable method. Formulation of cells with standard
pharmaceutically acceptable carriers and/or excipients may be
carried out using routine methods in the pharmaceutical art. The
exact nature of a formulation will depend upon several factors
including the cells to be administered and the desired route of
administration. Suitable types of formulation are fully described
in Remington's Pharmaceutical Sciences, 19.sup.th Edition, Mack
Publishing Company, Eastern Pennsylvania, USA.
[0164] The cells may be administered by any route. Suitable routes
include, but are not limited to, intravenous, intramuscular,
intraperitoneal or other appropriate administration routes. If the
damaged tissue is cardiac tissue, the cells may be administered via
an endomyocardial, epimyocardial, intraventicular, intracoronary,
retrograde coronary sinus, intra-arterial, intra-pericardial or
intravenous route. If the damaged tissue is bone, the cells may be
administered via an intraosseous route or to the site of the
injury, such as a fracture, or disease. If the damaged tissue is
cartilage, tendon, ligament, liver, kidney or lung tissue, the
cells may be administered directly into the tissue. If the damaged
tissue is lung tissue, the cells may be introduced via an
intra-pulmonary route. If the damaged tissue is liver or kidney,
the cells may be introduced via an intra-peritoneal route. The
cells are preferably administered intravenously.
[0165] Compositions may be prepared together with a physiologically
acceptable carrier or diluent. Typically, such compositions are
prepared as liquid suspensions of cells. The cells may be mixed
with an excipient which is pharmaceutically acceptable and
compatible with the active ingredient. Suitable excipients are, for
example, water, saline, dextrose, glycerol, of the like and
combinations thereof.
[0166] In addition, if desired, the pharmaceutical compositions of
the invention may contain minor amounts of auxiliary substances
such as wetting or emulsifying agents, pH buffering agents, and/or
adjuvants which enhance effectiveness. The composition preferably
comprises human serum albumin.
[0167] One suitable carrier or diluents is Plasma-Lyte A.RTM.. This
is a sterile, nonpyrogenic isotonic solution for intravenous
administration. Each 100 mL contains 526 mg of Sodium Chloride, USP
(NaCl); 502 mg of Sodium Gluconate (C6H11NaO7); 368 mg of Sodium
Acetate Trihydrate, USP (C2H3NaO2.3H2O); 37 mg of Potassium
Chloride, USP (KCl); and 30 mg of Magnesium Chloride, USP
(MgCl2.6H2O). It contains no antimicrobial agents. The pH is
adjusted with sodium hydroxide. The pH is 7.4 (6.5 to 8.0).
[0168] The IMP cells may be contained within one or more liposomes
and/or one or more microbubbles. Suitable liposomes are known in
the art. Suitable liposomes are disclosed in, for example,
Akbarzadeh et al. Nanoscale Research Letters 2013, 8:102 and
Meghana et al. International Journal Of Pharmaceutical And Chemical
Sciences, 2012, 1(1): 1-10. Suitable lipids for use in forming
liposomes are discussed below with reference to microbubbles.
[0169] Microbubbles, their formation and biomedical uses are known
in the art (e.g. Sirsi and Borden, Bubble Sci Eng Technol. November
2009; 1(1-2): 3-17). Microbubbles are bubbles smaller than one
millimetre in diameter and larger than one micrometre in diameter.
The microbubble used in the present invention is preferably 8 .mu.m
or less in diameter, such as 7 .mu.m or less in diameter, 6 .mu.m
or less in diameter, 5 .mu.m or less in diameter, 4 .mu.m or less
in diameter, 3 .mu.m or less in diameter or 2 .mu.m or less in
diameter.
[0170] The microbubble may be formed from any substance. The
general composition of a microbubble is a gas core stabilised by a
shell. The gas core may comprise air or a heavy gas, such as
perfluorocarbon, nitrogen or perflouropropane. Heavy gases are less
water soluble and so are less likely to leak out from the
microbubble leading to microbubble dissolution. Microbubbles with
heavy gas cores typically last longer in circulation.
[0171] The shell may be formed from any material. The shell
material preferably comprises a protein, a surfactant, a lipid, a
polymer or a mixture thereof.
[0172] Suitable proteins, include but are not limited to, albumin,
lysozyme and avidin. Proteins within the shell may be
chemically-crosslinked, for instance by cysteine-cysteine linkage.
Other crosslinkages are known in the art.
[0173] Suitable surfactants include, but are not limited to,
sorbitan monopalmitate (such as SPAN-40), polysorbate detergents
(such as TWEEN-40), mixtures of SPAN-40 and TWEEN-40 and sucrose
stearate (mono- and di-ester).
[0174] Suitable polymers include, but are not limited to, alginate
polymers, double ester polymers of ethylidene, the copolymer
poly(D,L-lactide-co-glycolide) (PLGA), poly(vinyl alcohol) (PVA),
the copolymer polyperfluorooctyloxycaronyl-poly(lactic acid)
(PLA-PFO) and other block copolymers. Block copolymers are
polymeric materials in which two or more monomer sub-units that are
polymerized together to create a single polymer chain. Block
copolymers typically have properties that are contributed by each
monomer sub-unit. However, a block copolymer may have unique
properties that polymers formed from the individual sub-units do
not possess. Block copolymers can be engineered such that one of
the monomer sub-units is hydrophobic (i.e. lipophilic), whilst the
other sub-unit(s) are hydrophilic whilst in aqueous media. In this
case, the block copolymer may possess amphiphilic properties and
may form a structure that mimics a biological membrane. The block
copolymer may be a diblock (consisting of two monomer sub-units),
but may also be constructed from more than two monomer sub-units to
form more complex arrangements that behave as amphipiles. The
copolymer may be a triblock, tetrablock or pentablock copolymer.
Block copolymers may also be constructed from sub-units that are
not classed as lipid sub-materials; for example a hydrophobic
polymer may be made from siloxane or other non-hydrocarbon based
monomers. The hydrophilic sub-section of block copolymer can also
possess low protein binding properties, which allows the creation
of a membrane that is highly resistant when exposed to raw
biological samples. This head group unit may also be derived from
non-classical lipid head-groups.
[0175] Any lipid material that forms a microbubble may be used. The
lipid composition is chosen such that the microbubble has the
required properties, such surface charge, packing density or
mechanical properties. The lipid composition can comprise one or
more different lipids. For instance, the lipid composition can
contain up to 100 lipids. The lipid composition preferably contains
1 to 10 lipids. The lipid composition may comprise
naturally-occurring lipids and/or artificial lipids.
[0176] The lipid typically comprises a head group, an interfacial
moiety and two hydrophobic tail groups which may be the same or
different. Suitable head groups include, but are not limited to,
neutral head groups, such as diacylglycerides (DG) and ceramides
(CM); zwitterionic head groups, such as phosphatidylcholine (PC),
phosphatidylethanolamine (PE) and sphingomyelin (SM); negatively
charged head groups, such as phosphatidylglycerol (PG);
phosphatidylserine (PS), phosphatidylinositol (PI), phosphatic acid
(PA) and cardiolipin (CA); and positively charged headgroups, such
as trimethylammonium-Propane (TAP). Suitable interfacial moieties
include, but are not limited to, naturally-occurring interfacial
moieties, such as glycerol-based or ceramide-based moieties.
Suitable hydrophobic tail groups include, but are not limited to,
saturated hydrocarbon chains, such as lauric acid (n-Dodecanolic
acid), myristic acid (n-Tetradecononic acid), palmitic acid
(n-Hexadecanoic acid), stearic acid (n-Octadecanoic) and arachidic
(n-Eicosanoic); unsaturated hydrocarbon chains, such as oleic acid
(cis-9-Octadecanoic); and branched hydrocarbon chains, such as
phytanoyl. The length of the chain and the position and number of
the double bonds in the unsaturated hydrocarbon chains can vary.
The length of the chains and the position and number of the
branches, such as methyl groups, in the branched hydrocarbon chains
can vary. The hydrophobic tail groups can be linked to the
interfacial moiety as an ether or an ester.
[0177] The lipids can also be chemically-modified. The head group
or the tail group of the lipids may be chemically-modified.
Suitable lipids whose head groups have been chemically-modified
include, but are not limited to, PEG-modified lipids, such as
1,2-Diacyl-sn-Glycero-3-Phosphoethanolamine-N-[Methoxy(Polyethylene
glycol)-2000]; functionalised PEG Lipids, such as
1,2-Distearoyl-sn-Glycero-3
Phosphoethanolamine-N-[Biotinyl(Polyethylene Glycol)2000]; and
lipids modified for conjugation, such as
1,2-Dioleoyl-sn-Glycero-3-Phosphoethanolamine-N-(succinyl) and
1,2-Dipalmitoyl-sn-Glycero-3-Phosphoethanolamine-N-(Biotinyl).
Suitable lipids whose tail groups have been chemically-modified
include, but are not limited to, polymerisable lipids, such as
1,2-bis(10,12-tricosadiynoyl)-sn-Glycero-3-Phosphocholine;
fluorinated lipids, such as
1-Palmitoyl-2-(16-Fluoropalmitoyl)-sn-Glycero-3-Phosphocholine;
deuterated lipids, such as
1,2-Dipalmitoyl-D62-sn-Glycero-3-Phosphocholine; and ether linked
lipids, such as 1,2-Di-O-phytanyl-sn-Glycero-3-Phosphocholine. The
lipids may be chemically-modified or functionalised to facilitate
coupling of the ligands, receptors ro antibodies as discussed
above.
[0178] The lipid composition may comprise one or more additives
that will affect the properties of the microbubble. Suitable
additives include, but are not limited to, fatty acids, such as
palmitic acid, myristic acid and oleic acid; fatty alcohols, such
as palmitic alcohol, myristic alcohol and oleic alcohol; sterols,
such as cholesterol, ergosterol, lanosterol, sitosterol and
stigmasterol; lysophospholipids, such as
1-Acyl-2-Hydroxy-sn-Glycero-3-Phosphocholine; and ceramides.
[0179] The microbubble shell is preferably formed from a
phospholipid. Suitable phospholipids are known in the art.
[0180] There are several commercially available lipid shell
microbubble formulations such as Definity (Lantheus Medical
Imaging) and Sonovue.RTM. (Bracco Diagnostics).
[0181] The microbubble may also be formed from a polymer-surfactant
hybrid that involves forming polyelectrolyte multilayer (PEM)
shells on a preformed microbubble. The preformed microbubble is
coated with a charged surfactant or protein layer, which serves as
a substrate for PEM deposition. The layer-by-layer assembly
technique is used to sequentially adsorb oppositely charged
polyions to the microbubble shell. For instance, PEM can be
deposited onto microbubbles using poly(allylamine hydrochloride)
(PAH) and poly(styrene sulfonate) (PSS) for the polyion pair. PEM
microbubbles with phospholipid containing the cationic headgroup
trimethylammonium propane (TAP) as the underlying shell and DNA and
poly(L-lysine) (PLL) as the polyion pair have also been
developed.
[0182] The microbubble is typically formed by providing an
interface between a gas and a microbubble shell material. Any of
the materials discussed above may be used. Some materials, such as
phospholipids, spontaneously form microbubbles. Phospholipids self
assemble into a microbubble. Other materials require sonication of
the interface, i.e. the application of sound energy or sonic waves
to the interface. Ultrasonic waves are typically used. Suitable
methods are known in the art for sonication.
[0183] The microbubble may be loaded with the IMP cells after
formation of the microbubble or during formation of the
microbubble.
[0184] The IMP cells are administered in a manner compatible with
the dosage formulation and in such amount will be therapeutically
effective. The quantity to be administered depends on the subject
to be treated, capacity of the subject's immune system and the
degree repair desired. Precise amounts of IMP cells required to be
administered may depend on the judgement of the practitioner and
may be peculiar to each subject.
[0185] Any suitable number of cells may be administered to a
subject. For example, at least, or about, 0.2.times.10.sup.6,
0.25.times.10.sup.6, 0.5.times.10.sup.6, 1.5.times.10.sup.6,
4.0.times.10.sup.6 or 5.0.times.10.sup.6 cells per kg of patient
may administered. For example, at least, or about, 10.sup.5,
10.sup.6, 10.sup.7, 10.sup.8, 10.sup.9 cells may be administered.
As a guide, the number of cells of the invention to be administered
may be from 10.sup.5 to 10.sup.9, preferably from 10.sup.6 to
10.sup.8. Typically, up to 2.times.10.sup.8 IMP cells are
administered to each patient. Any of the specific numbers discussed
above with reference to the populations of the invention may be
administered. In such cases where cells are administered or
present, culture medium may be present to facilitate the survival
of the cells. In some cases the cells of the invention may be
provided in frozen aliquots and substances such as DMSO may be
present to facilitate survival during freezing. Such frozen cells
will typically be thawed and then placed in a buffer or medium
either for maintenance or for administration.
Hybrid Composition
[0186] One or more IMP cells of the invention may form part of a
hybrid composition as disclosed in the UK Application being filed
concurrently with this application (CTL Ref: FIBRE1) and are
preferably administered to a patient as part of such a composition.
In particular, the invention provides a hybrid composition, which
comprises:
[0187] (a) one or more biocompatible fibres;
[0188] (b) one or more IMP cells of the invention; and
[0189] (c) one or more biocompatible components which (i) attach
the one or more therapeutic cells to the one or more fibres and/or
embed the one or more therapeutic cells and the one or more fibres
and/or (ii) are capable of attaching the composition to a
tissue.
[0190] The hybrid composition of the invention comprises one or
more biocompatible fibres. A fibre is biocompatible if it does not
cause any adverse reactions or side effects when contacted with a
damaged tissue.
[0191] Any number of biocompatible fibres may be present in the
composition. The composition may comprise only one fibre. The
composition typically comprises more than one fibre, such at least
2, at least 5, at least 10, at least 20, at least 30, at least 40,
at least 50, at least 100, at least 200, at least 500 fibres, at
least 1000 fibres or even more fibres.
[0192] Suitable biocompatible fibres are known in the art. The one
or more biocompatible fibres may be natural or synthetic. Preferred
biocompatible fibres include, but are not limited to, cellulose
fibres, collagen fibres, collagen-glycosaminoglycan fibres, gelatin
fibres, silk fibroin fibres, one or more fibrin fibres, chitosan
fibres, starch fibres, alginate fibres, hyaluronan fibres,
poloaxmer fibres or a combination thereof. The glycosaminoglycan is
preferably chondroitin. The cellulose is preferably
carboxymethylcellulose, hydroxypropylmethylcellulose or
methylcellulose. The poloaxmer is preferably pluronic acid,
optionally Pluronic F-127.
[0193] If more than one fibre is present in the composition, the
population of fibres may be homogenous. In other words, all of the
fibres in the population may be the same type of fibre, e.g.
cellulose fibres. Alternatively, the population of fibres may be
heterogeneous. In other words, the population of fibres may contain
different types of fibre, such cellulose fibres and collagen
fibres.
[0194] The one or more fibres may be any length. The one or more
fibres are preferably approximately the same length as the depth of
the damage in the tissue which is to be treated using the
composition. The length of one or more fibres is preferably
designed such that the composition can penetrate a damaged tissue
to a prescribed depth. The one or more fibres may be any length.
The lower limit of the length of the one or more fibres is
typically determined by the diameter of the one or more therapeutic
cells. Suitable lengths include, but are not limited to, at least 1
.mu.m in length, at least 10 .mu.m in length, at least 100 .mu.m in
length, at least 500 .mu.m in length, at least 1 mm in length, at
least 10 mm (1 cm) in length, at least 100 mm (10 cm) in length, at
least 500 mm (50 cm) in length or at least 1000 mm (100 cm or 1 m)
in length. The one or more fibres may be even longer. For instance,
the one or more fibres may be up to 5m or 10m in length, for
instance if being used to repair damage along the human intestinal
tract, or even longer if being used in larger animals, such as
horses. The length of the one or more fibres is typically
determined by their intended use and/or their ability to be
manipulated, for instance by a surgeon, by a robot or via some
other means, such as magnetically.
[0195] The one or more fibres may be charged. The one or more
fibres are preferably positively-charged. The one or more fibres
are preferably negatively-charged.
[0196] The one or more fibres may be magnetic. The one or more
fibres may be modified to include one or more magnetic atoms or
groups. This allows magnetic targeting of the composition. The
magnetic atoms or groups may be paramagnetic or superparamagnetic.
Suitable atoms or groups include, but are not limited to, gold
atoms, iron atoms, cobalt atoms, nickel atoms and a metal chelating
groups, such as nitrilotriacetic acid, containing any of these
atoms. The metal chelating group may, for instance, comprise a
group selected from --C(.dbd.O)O--, --C--O--C, C(.dbd.O), --NH--,
--C(.dbd.O)--NH, --C(.dbd.O)--CH.sub.2--I, --S(.dbd.O).sub.2-- and
--S--.
[0197] The composition also comprises one or more biocompatible
components. The one or more biocompatible components (i) attach the
one or more therapeutic cells to the one or more fibres and/or
embed the one or more therapeutic cells and the one or more fibres
and/or (ii) are capable of attaching the composition to a tissue.
The one or more biocompatible components may (a) attach the one or
more therapeutic cells to the one or more fibres, (b) embed the one
or more therapeutic cells and the one or more fibres, (c) be
capable of attaching the composition to a tissue, (d) attach the
one or more therapeutic cells to the one or more fibres and embed
the one or more therapeutic cells and the one or more fibres, (e)
attach the one or more therapeutic cells to the one or more fibres
and be capable of attaching the composition to a tissue, (f) embed
the one or more therapeutic cells and the one or more fibres and be
capable of attaching the composition to a tissue or (g) attach the
one or more therapeutic cells to the one or more fibres, embed the
one or more therapeutic cells and the one or more fibres and be
capable of attaching the composition to a tissue.
[0198] A component is biocompatible if it does not cause any
adverse reactions or side effects when contacted with a damaged
tissue.
[0199] Any number of biocompatible components may be present in the
composition. The composition typically comprises only one component
or two components. The composition may comprise more than two
components, such as at least 3, at least 5, at least 10, at least
20, at least 30, at least 40, at least 50 components or even more
components.
[0200] The one or more biocompatible components preferably comprise
a biocompatible adhesive which attaches the one or more therapeutic
cells to the one or more fibres. The biocompatible adhesive may
attach the one or more therapeutic cells (a) on the surface of the
one or more fibres, (b) within the one or more fibres or (c) both
on the surface of and within the one or more fibres.
[0201] The biocompatible adhesive may be natural or synthetic.
Suitable biocompatible adhesives are known in the art. Suitable
adhesives include, but are not limited to, fibrin, fibrin gel,
integrin, integrin gel, cadherin and cadherin gel.
[0202] The one or more biocompatible components preferably comprise
a biocompatible gel which embeds the one or more therapeutic cells
and the one or more fibres. Suitable biocompatible gels are known
in the art. The biocompatible gel may be natural or synthetic.
Preferred biocompatible gels include, but are not limited to, a
cellulose gel, a collagen gel, a gelatin gel, a fibrin gel, a
chitosan gel, a starch gel, an alginate gel, a hyaluronan gel, an
agarose gel, a poloaxmer gel or a combination thereof.
[0203] The cellulose gel may be formed from any of the celluloses
discussed above. The cellulose polymer concentration is preferably
from about 1.5% (w/w) to about 4.0% (w/w), such as from about 2.0%
(w/w) to about 3.0% (w/w). The cellulose polymer preferably has a
molecular weight of from about 450,000 to about 4,000,000, such as
from about 500,000 to about 3,500,000, from about 500,000 to about
3,000,000 or from about 750,000 to about 2,500,000 or from about
1000,000 to about 2,000,000.
[0204] The poloaxmer gel is preferably a pluronic acid gel,
optionally a Pluronic F-127 gel. The adhesive and/or gel preferably
has a viscosity in the range of 1000 to 500,000 mPas (cps) at room
temperature, such as from about 1500 to about 450,000 mPas at room
temperature, from about 2000 to about 400,000 mPas at room
temperature, from about 2500 to about 350,000 mPas at room
temperature, from about 5000 to about 300,000 mPas at room
temperature, from about 10,000 to about 250,000 mPas at room
temperature, from about 50,000 to about 200,000 mPas at room
temperature or from about 50,000 to about 150,000 mPas at room
temperature.
[0205] Viscosity is a measure of the resistance of the adhesive
and/or gel to being deformed by either shear stress or tensile
stress. Viscosity can be measured using any method known in the
art. Suitable methods include, but are not limited to, using a
viscometer or a rheometer.
[0206] Room temperature is typically from about 18.degree. C. to
about 25.degree. C., such as from about 19.degree. C. to about
24.degree. C. or from about 20.degree. C. to about 23.degree. C. or
from about 21.degree. C. to about 22.degree. C. Room temperature is
preferably any of 18.degree. C., 19.degree. C., 20.degree. C.,
21.degree. C., 22.degree. C., 23.degree. C., 24.degree. C. and
25.degree. C. Viscosity is most preferably measured at 25.degree.
C.
[0207] The one or more biocompatible components preferably
comprises a biocompatible adhesive which attaches the one or more
therapeutic cells to the one or more fibres and a biocompatible gel
which embeds the one or more therapeutic cells and the one or more
fibres. For instance, the composition may comprise a fibrin gel
which attaches the one or more therapeutic cells to the one or more
fibres and a cellulose gel which embeds the one or more therapeutic
cells and the one or more fibres.
[0208] In any of the embodiments discussed above, the biocompatible
adhesive and/or the biocompatible gel preferably comprises platelet
lysate. For instance, the adhesive and/or the gel may be a platelet
lystae gel. Platelet lysate refers to the combination of natural
growth factors contained in platelets that has been released
through lysing those platelets. Lysis can be accomplished through
chemical means (i.e. CaCl.sub.2), osmotic means (use of distilled
H.sub.2O) or through freezing/thawing procedures. Platelet lysate
can be derived from whole blood as described in U.S. Pat. No.
5,198,357. Platelet lysate is preferably prepared as described in
PCT/GB12/052911 (published as WO 2013/076507). For instance, it may
be prepared by subjecting a population of platelets to at least one
freeze-thaw cycle, wherein the freeze portion of each cycle is
carried out at a temperature lower than or equal to -78.degree.
C.
[0209] The adhesive and/or gel preferably comprises (a) platelet
lysate, (b) at least one pharmaceutically acceptable polymer and
(c) at least one pharmaceutically acceptable positively charged
chemical species selected from the group consisting of lysine,
arginine, histidine, aspartic acid, glutamic acid, alanine,
methionine, proline, serine, asparagine, cysteine, polyamino acids,
protamine, aminoguanidine, zinc ions and magnesium ions, wherein
the composition is an aqueous gel having a viscosity in the range
of 1000 to 500,000 mPas (cps) at room temperature. The
pharmaceutically acceptable polymer is preferably cellulose or a
poloaxmer. It may be any of the celluloses and poloaxmers discussed
above.
[0210] The platelet lysate is preferably human platelet lysate.
Platelet lysate is discussed in more detail above.
[0211] The hybrid composition may be contained within one or more
liposomes or one or more microbubbles. Such structures are known in
the art.
[0212] The following Examples illustrate the invention.
EXAMPLES
Example 1--Bone Marrow and Peripheral Blood Isolation &
Expansion of IMP Cells
[0213] A bone marrow sample was diluted with Hank Buffered Saline
Solution and layered over Ficoll-Paque for the isolation of
mononuclear cells (MCs) by centrifugation. The MCs were then
re-suspended in Hank Buffered Saline Solution and counted using
0.4% trypan blue exclusion assay to assess cellular viability.
Cells were seeded in T25 flasks (in 5 ml of cell culture media,
.alpha.MEM, GlutaMAX, penicillin-streptomycin, platelet lysate,
heparin), and incubated at 37.degree. C., 5% CO2. On day 8 the
media was changed. Cells were monitored daily for observation of
IMP-like cells and, if present, harvested using cell dissociating
solution according to manufacturer's instructions and sub-cultured
in the same media as above. Cells were cryopreserved in passage 2
in culture media supplemented with 10% dimethyl sulfoxide to
-80.degree. C. and stored in liquid nitrogen for later use.
Example 2--HT-FACS Analysis
[0214] High-throughput fluorescence activated cell sorting
(HT-FACS) analysis is a high-throughput screening platform which
can rapidly characterize the cell surface phenotype of cells in
suspension, with over 370 cell surface markers currently in the
panel. This platform has undergone extensive validation and has
been performed on many types of human tissues and cells. The panel
consists of 375 human cell surface-specific antibodies arrayed in
96-well plates.
[0215] The aim was to determine the surface antigen expression
profile of human IMPs of the invention and human MSCs obtained from
Lonza.RTM.. The high-throughput-FACS (HT-FACS) platform allows the
screening of 375 surface antigens.
[0216] One vial of cryopreserved PB-MSCs (1.times.106 cells/ml) was
seeded in a T75 cm2 flask containing 15 mL of CTL media (37.degree.
C., 5% CO2). Cells were grown until confluence of 80-90% changing
the media every 2-3 days. To passage the cells, the media was
removed and cells were washed twice with PBS. Cells were treated
with 3 ml of Trypsin 0.25% until detached. Eight ml of media were
added to inactivate the trypsin and cells were collected by
centrifugation at 400 g for 5 min. Cells were re-suspended in 5 ml
of media and seeded in a T175 cm2 flask containing 30 mL of CTL
media (37.degree. C., 5% CO2). Between 8 to 10 T175 cm2 flasks at
80-90% confluence were required to harvest 20-30 million cells (at
passage 4) for the HT-FACS screening. In order to obtain a
sufficient number of flow cytometry "events" per antibody,
approximately 20 million viable cells is optimal. To collect the
cells, the media was removed and cells were washed twice with PBS.
Cells were treated with 5 ml of Trypsin 0.25% until detached. Media
was added (8 ml) to inactivate the trypsin and collect the cells.
Cells were centrifuged at 400 g for 5 min. The cell pellets were
re-suspended (single-cell suspension) in 5 ml total of HBSS (Hank's
Balanced Salt Solution minus calcium/magnesium, supplemented with 2
mM EDTA and 1% BSA). One aliquot of the sample (10 .mu.l) was used
to determine the total number of viable cells by using exclusion
dye (0.2% trypan blue).
[0217] 100 .mu.l of sample were loaded into each well (about 40,000
cells per well assuring the collection of 10,000 to 20,000 events
in the FACS). The samples were run in a BD FACSDiva upgraded with a
BD High Throughput Sampler (automated sampler). The analysis of
flow cytometry data were performed using FlowJo Software. The
results were provided in plots, and an Excel spreadsheet containing
the percentage of positive cells and median fluorescence intensity
(MFI) for each antibody.
TABLE-US-00001 TABLE 1 Results of the HT-FACS analysis % Lonza
.RTM. # Marker Alternative name: % IMP MSC 1 BLTR-1 6.7 1.37 2 B2-
99.8 100 microglobulin 3 CA9 Carbonic anhydrase 9 5.22 0 4 CDH3
Cadherin-3/P-Cadherin 2.93 0.475 5 CDH6 Cadherin-6 0.6 0.235 6
CDH11 Cadherin-11 61.6 0.88 7 CDw93 11.5 4.75 8 CDw198 CCR8 10.6
5.17 9 CDw199 CCR9 17.2 2.54 10 CDw210 Interleukin 10 receptor,
alpha subunit (IL10RA) 10.8 0.622 11 CDw218a interleukin-18
receptor 1 (IL18R1) 0.384 0 12 CDw329 Sialic acid-binding Ig-like
lectin 9 (Siglec 9) 0.182 0 13 CD1a 0.338 0.28 14 CD1b 0.766 0.745
15 CD1c 15.7 0.926 16 CD1d R3G1 2.7 0 17 CD2 LFA-2 0.292 0.526 18
CD3 0.158 0 19 CD3e 0.087 0 20 CD4 1.11 0.157 21 CD5 Leu-1 0.151
0.34 22 CD6 1.04 2.68 23 CD7 GP40/Leu-9 0.239 0.24 24 CD8 0.214 0
25 CD8b 4.34 0.705 26 CD9 BTCC-1 38.1 51.9 27 CD10 Neprilysin
(NEP)/common acute lymphoblastic 90.6 87.1 leukemia antigen (CALLA)
28 CD11a ITGAL, LFA-1 1.57 0 29 CD11b Integrin alpha M (ITGAM) 6.24
0 30 CD11c Integrin, alpha X (ITGAX) 1.8 0 31 CD13 Alanine
aminopeptidase (ANPEP) 100 100 32 CD14 8.03 6.25 33 CD15 SSEA-1
0.137 0.474 34 CD16 Fc Receptor 10.1 3.73 35 CD16b Fc fragment of
IgG, low affinity IIIb, receptor 0.331 0 (FCGR3B) 36 CD17
Lactosylceramide (LacCer) 20.9 0.462 37 CD18 Integrin beta-2 0.65 0
38 CD19 0.21 0 39 CD20 0.176 0 40 CD21 Complement receptor type 2
(Cr2)/Epstein-Barr 0.66 0 virus receptor (EBV R) 41 CD22
BL-CAM/Siglec-2 0.596 0 42 CD23 Low affinity immunoglobulin epsilon
Fc receptor 0.551 0.234 (FCER2) 43 CD24 0.987 4 44 CD25
Interleukin-2 receptor subunit alpha (IL2RA) 1.44 1.67 45 CD26
Dipeptidyl peptidase IV (DPP4) 21.3 6.33 46 CD27 Tumor necrosis
factor receptor superfamily 0.409 0 member 7 (TNFRSF7) 47 CD28
0.643 0 48 CD29 Integrin beta-1 (ITGB1) 100 100 49 CD30 Tumor
necrosis factor receptor superfamily 0.446 0 member 8 (TNFRSF8) 50
CD31 PECAM 1.29 0.214 51 CD32 Low affinity immunoglobulin gamma Fc
region 0.698 3.46 receptor II-b 52 CD33 Siglec-3 1.25 0.372 53 CD34
0.287 0.885 54 CD35 Complement receptor type 1 (Cr1) 0.134 0 55
CD36 Platelet glycoprotein 4/Thrombospondin receptor 0.458 3.57 56
CD37 Tetraspanin-26 (TSPAN26) 0.0917 0.182 57 CD38 ADP-ribosyl
cyclase 1 0.28 0 58 CD39 Ectonucleoside triphosphate
diphosphohydrolase 0.126 21.8 NTPdase 1 59 CD40 Tumor necrosis
factor receptor superfamily 0.132 3.12 member 5 (TNFRSF5) 60 CD41a
0.293 0 61 CD41b 0.075 0 62 CD42a Platelet glycoprotein IX 0.528
0.131 63 CD42b Platelet glycoprotein Ib alpha chain 7.29 0 64 CD43
Leukosialin 0.406 1.81 65 CD44 Epican 99.9 99.7 66 CD45
Receptor-type tyrosine-protein phosphatase C, 0.271 0 Leukocyte
common antigen 67 CD45RA 5.18 2.99 68 CD45RB 0.283 0.671 69 CD45RO
0.57 0 70 CD46 Membrane cofactor protein, Trophoblast 78.1 22.5
leukocyte common antigen 71 CD47 Antigenic surface determinant
protein OA3 92.3 99.9 72 CD48 SLAM F2 0.141 0.125 73 CD49a Integrin
alpha-1 (ITGA1) 24 51.5 74 CD49b Integrin alpha-2 (ITGA2) 97.7 45.8
75 CD49c Integrin alpha-3 (ITGA3) 99.9 99.6 76 CD49d Integrin
alpha-4 (ITGA4) 93.7 26 77 CD49e Integrin alpha-5 (ITGA5) 100 99.8
78 CD49f Integrin alpha-6 (ITGA6) 93.3 24.1 79 CD50 ICAM-3 0.244
0.8 80 CD51/CD61 92.7 68 81 CD52 CAMPATH-1 antigen 0.218 0.128 82
CD53 1.66 0.292 83 CD54 ICAM-1 23.1 23.7 84 CD55 Complement
decay-accelerating factor 94.5 52.5 85 CD56 NCAM 3.05 4.71 86 CD57
Killer cell lectin-like receptor subfamily G 0.193 0 member 1 87
CD58 LFA-3 99.7 98.1 88 CD59 Protectin 100 100 89 CD60b 34 10.9 90
CD61 Integrin beta-3 ITGB3 81.8 56.7 91 CD62E E-Selectin Ligand
2.33 1.03 92 CD62L L-Selectin Ligand 0.432 0.151 93 CD62P
P-Selectin Ligand 0.325 0.924 94 CD63 Lysosomal-associated membrane
protein 3 99.1 95.8 (LAMP-3) 95 CD64 High affinity immunoglobulin
gamma Fc receptor 0.263 0.225 I (Fc-gamma RI) 96 CD65 0.825 0 97
CD65s 7.62 0.539 98 CD66 Pregnancy-specific beta-1-glycoprotein 1
PSGB1 0.474 0.737 99 CD66b 0.129 0 100 CD66c Carcinoembryonic
antigen-related cell adhesion 23.4 7.33 molecule 6 101 CD66d
Carcinoembryonic antigen-related cell adhesion 2.06 0.322 molecule
3 102 CD66e Carcinoembryonic antigen-related cell adhesion 56.1
13.6 molecule 5 103 CD69 Activation inducer molecule (AIM) 0.296
0.279 104 CD70 Tumor necrosis factor ligand superfamily 0.36 0.187
member 7 (TNFSF7) 105 CD71 Transferrin receptor protein 1 51 4.71
106 CD72 0.036 0.334 107 CD73 5'-nucleotidase/SH3/SH4 100 99.8 108
CD74 HLA class II histocompatibility antigen gamma 0.177 0.587
chain 109 CD75 Beta-galactoside alpha-2,6-sialyltransferase 1
0.0789 0.304 110 CD77 Lactosylceramide
4-alpha-galactosyltransferase 7.15 2.4 111 CD79a B-cell antigen
receptor complex-associated 15.4 0.45 protein alpha chain 112 CD79b
B-cell antigen receptor complex-associated 4.87 0.317 protein beta
chain 113 CD80 Activation B7-1 antigen 2.94 4.57 114 CD81
Tetraspanin-28 100 99.9 115 CD82 Tetraspanin-27 96.3 82.7 116 CD83
27.9 1.34 117 CD84 SLAM F5 7.94 4.1 118 CD85a Leukocyte
immunoglobulin-like receptor 6.76 0.971 subfamily B member 3
(LIR-3) 119 CD85d Leukocyte immunoglobulin-like receptor 17 0.98
subfamily B member 3 (LIR-2) 120 CD85g Leukocyte
immunoglobulin-like receptor 47.2 6.15 subfamily A member 4 121
CD85h Leukocyte immunoglobulin-like receptor 15.6 0 subfamily A
member 2 (LILRA2) 122 CD85j Leukocyte immunoglobulin-like receptor
20.6 0.221 subfamily B member 1 (LIR-1) 123 CD86 24.7 0.702 124
CD87 Urokinase plasminogen activator surface receptor 0.178 1.61
(uPAR) 125 CD88 C5a anaphylatoxin chemotactic receptor 1 1.32 0.352
126 CD89 Immunoglobulin alpha Fc receptor 5.73 0.244 127 CD90 Thy-1
membrane glycoprotein 100 99.3 128 CD91 Prolow-density lipoprotein
receptor-related 95.5 63.4 protein 1 (LRP-1) 129 CD92 Choline
transporter-like protein 1 35.4 33.3 130 CD94 Natural killer cells
antigen CD94 KLRD1 0.121 0.321 131 CD95 CD95L (Ligand)/Tumor
necrosis factor ligand 98.9 66.7 superfamily member 6 (TNFSF6) 132
CD96 T-cell surface protein tactile 21 2.63 133 CD97 1.64 0.434 134
CD98 Large neutral amino acids transporter small 100 99.9 subunit 1
135 CD99 T-cell surface glycoprotein E2 24.8 0.224 136 CD100
Semaphorin-4D 0.103 0.132 137 CD101 Immunoglobulin superfamily
member 2 (IgSF2) 0.29 0 138 CD102 ICAM-2 9.24 2.91 139 CD103
Integrin alpha-E (ITGAE) 0.152 0.297 140 CD104 Integrin beta-4
(ITGB4) 4.06 99.3 141 CD105 Endoglin (SH2) 99.9 100 142 CD106 VCAM
6.93 4.64 143 CD107a Lysosome-associated membrane glycoprotein 1
0.717 0.337 (LAMP-1) 144 CD107b Lysosome-associated membrane
glycoprotein 2 0.221 0.225 (LAMP-2) 145 CD108 Semaphorin-7A 99.7 78
146 CD109 1.89 0.253 147 CD110 Thrombopoietin receptor (TPO-R) 55.6
16.6 148 CD111 Herpes virus entry mediator C 90.7 0 149 CD112
Poliovirus receptor-related protein 2 12.1 0.64 150 CD114
Granulocyte colony-stimulating factor receptor 54.9 4.83
(GCSFR/CSF3R) 151 CD115 Macrophage colony-stimulating factor 1
receptor 8.41 0 CSF-1 receptor (CSF-1-R) 152 CD116
Granulocyte-macrophage colony-stimulating 17 2.61 factor receptor
subunit alpha GM-CSF-R-alpha 153 CD117 Mast/stem cell growth factor
receptor Kit (c-kit) 31.5 2.56 154 CD118 Leukemia inhibitory factor
receptor (LIF-R) 67.4 0 155 CD119 Interferon gamma receptor 1
(IFNgammaR) 78.5 24.8 156 CD120a Tumor necrosis factor receptor
superfamily 38.1 0 member 1A (TNFR1) 157 CD120b Tumor necrosis
factor receptor superfamily 1.11 0.297 member 1B (TNFR2) 158 CD121b
Interleukin-1 receptor type 2 (IL1R2) 39.8 2.75 159 CD122
Interleukin-2 receptor subunit beta (IL2RB) 41.7 4.56 160 CD123
Interleukin-3 receptor subunit alpha (IL3RA) 46.9 7.06 161 CD124
Interleukin-4 receptor subunit alpha IL4RA) 1.52 0.225 162 CD125
Interleukin-5 receptor subunit alpha (IL5RA) 19.5 0 163 CD126
Interleukin-6 receptor subunit alpha (IL-6R 1) 7.05 0.709 164 CD127
Interleukin-7 receptor subunit alpha (IL7RA) 18.5 12.5 165 CD129
Interleukin-9 receptor (IL9R) 0.178 0 166 CD130 Interleukin-6
receptor subunit beta (IL6ST) 83.6 8.15 167 CD131 Cytokine receptor
common subunit beta 0.684 0 168 CD132 Cytokine receptor common
subunit gamma 78.8 3.43 (IL2RG) 169 CD133 AC-133 (Prominin-1) 0.054
0 170 CD134 Tumor necrosis factor receptor superfamily 8.15 1.29
member 4 (TNFSF4) 171 CD135 Receptor-type tyrosine-protein kinase
FLT3 5.18 0.575 172 CD136 Macrophage-stimulating protein receptor
(MSP- 0.302 0 R) 173 CD137 Tumor necrosis factor receptor
superfamily 0.392 0 member 9 (TNFRSF9) 174 CD137L Mouse? 13.5 15.6
175 CD138 Syndecan-1 (SYND1) 0.227 0 176 CD140a Platelet-derived
growth factor receptor alpha 4.1 0.98 (PDGFRA) 177 CD140b
Platelet-derived growth factor receptor beta 89.1 97.8 (PDGFRB) 178
CD141 Thrombomodulin 21 0.385 179 CD142 Tissue
factor/Thromboplastin 0.478 0.555 180 CD143 Angiotensin-converting
enzyme (ACE) 29.3 0 181 CD144 Cadherin-5 0.0728 0.159 182 CD146
MUC18 94.2 89.5 183 CD147 Basigin 100 100 184 CD148 Receptor-type
tyrosine-protein phosphatase eta 84.6 0 185 CD150 Signaling
lymphocytic activation molecule 0.467 0.364 (SLAMF-1) 186 CD151
PETA-3 100 99.9 187 CD152 Cytotoxic T-lymphocyte protein 4 (CTLA-4)
6.45 5.87 188 CD153 Tumor necrosis factor ligand superfamily 10.9
1.19 member 8 (TNFSF8) 189 CD154 CD40 Ligand 0.357 0.893 190 CD155
Poliovirus receptor (PVR) 99.8 100 191 CD156b Disintegrin and
metalloproteinase domain- 81 36.4 containing protein 17 (ADAM-17)
192 CD157 ADP-ribosyl cyclase 2/Bone Marrow Stromal 0.713 6.33
Antigen 1 (BST-1) 193 CD158a Killer cell immunoglobulin-like
receptor 2DL1 0.0919 0.22 194 CD158b Killer cell
immunoglobulin-like receptor 2DL2 0.129 0.195 195 CD158b2 Killer
cell immunoglobulin-like receptor 2DL3 2.54 0
196 CD158d Killer cell immunoglobulin-like receptor 2DL4 56.3 1.56
197 CD158e2 Killer cell immunoglobulin-like receptor 3DL1 0.254 0
198 CD158f Killer cell immunoglobulin-like receptor 2DL5A 25 0 199
CD158i Killer cell immunoglobulin-like receptor 2DS4 21.9 3.12 200
CD159a NKG2-A/NKG2-B type II integral membrane 6.57 0.462 protein
(KLR-C1) 201 CD159c NKG2-C type II integral membrane protein 2.44
0.917 (KLR-C2) 202 CD160 1.07 0.9 203 CD161 Killer cell lectin-like
receptor subfamily B 5.95 3.64 member 1 (KLRB1) 204 CD162
P-selectin glycoprotein ligand 1 (PSGL-1) 13.2 4.41 205 CD163
Scavenger receptor cysteine-rich type 1 protein 0.197 0 M130 206
CD164 Sialomucin core protein 24 (MUC-24) 11.9 27 207 CD165 0.716
3.55 208 CD166 Activated leukocyte cell adhesion molecule 99.9 99.8
209 CD167 Discoidin domain-containing receptor 2 (DDR2) 0.496 7.69
210 CD169 Sialoadhesin/Siglec-1 1.76 0.178 211 CD170 Sialic
acid-binding Ig-like lectin 5 (Siglec-5) 11.9 74.3 212 CD171 Neural
cell adhesion molecule L1 (NCAM-L1) 1.9 0 213 CD172a
Tyrosine-protein phosphatase non-receptor type 61.8 3.33 substrate
1 (SHP-1) 214 CD172b Signal-regulatory protein beta-1 (SIRP-beta-1)
0.0955 0.285 215 CD172g Signal-regulatory protein gamma
(SIRP-gamma) 14.5 7.14 216 CD175s 96.2 27.1 217 CD177 Human
neutrophil alloantigen 2a (HNA-2a) 0.477 0.46 218 CD178 CD95L
(Ligand)/Tumor necrosis factor ligand 51.6 0.49 superfamily member
6 (TNFSF6) 219 CD179a 6.31 1.84 220 CD180 0.824 0.478 221 CD181
CXCR1 85 2.55 222 CD182 CXCR2 68.8 4.31 223 CD183 CXCR3 3.08 0 224
CD184 CXCR4 0.219 0.775 225 CD185 CXCR5 6.04 1.39 226 CD186 CXCR6
1.48 41.5 227 CD191 CCR1 12.6 0 228 CD192 CCR2 0.0662 0.0497 229
CD193 CCR3 51 8.16 230 CD194 CCR4 7.13 0 231 CD195 CCR5 1.02 1.94
232 CD196 CCR6 46.3 2.8 233 CD197 CCR7 0.159 0 234 CD200 OX-2
membrane glycoprotein (MOX-1)/(MOX- 0.594 0.912 2) 235 CD201
Endothelial protein C receptor 55.7 0.858 236 CD202b Angiopoietin-1
receptor TIE2/TEK 82.7 23.2 237 CD203c Ectonucleotide 8.66 0
pyrophosphatase/phosphodiesterase family member 3 (ENPP3) 238 CD204
Macrophage scavenger receptor types I and II 13.7 1.44 (MSR1) 239
CD205 Lymphocyte antigen 75 (Ly-75) 4.94 0 240 CD206 Macrophage
mannose receptor 1 (MMR) 0.205 0 241 CD207 C-type lectin domain
family 4 member K 0.0679 2.7 (Langerin) 242 CD208
Lysosome-associated membrane glycoprotein 3 3.27 0 (LAMP-3) 243
CD209 0.153 0 244 CD212 Interleukin-12 receptor subunit beta-1
(IL12RB1) 0.476 0.127 245 CD213a2 Interleukin-13 receptor subunit
alpha-2 8.7 8 (IL13RA2) 246 CD215 Interleukin-15 receptor subunit
alpha 14.6 0.86 247 CD217 Interleukin-17 receptor A (IL17RA) 29.8
35.8 248 CD218b Interleukin-18 receptor accessory protein (IL-18
23.4 0.463 R-beta) 249 CD220 Insulin Receptor IR 2.93 1.5 250 CD221
Insulin-like growth factor 1 receptor IGF-1R 3.16 1.1 251 CD222
Insulin-like growth factor 2 receptor IGF-2R 8.09 0.768 252 CD223
Lymphocyte activation gene 3 protein (LAG-3) 38.9 0 253 CD226 DNAX
accessory molecule 1 (DNAM-1) 1.15 0.22 254 CD227 Mucin-1 (MUC-1)
4.87 5.79 255 CD229 T-lymphocyte surface antigen Ly-9 0.579 5.56
256 CD230 Major prion protein (PrP) 99.9 100 257 CD231
Tetraspanin-7 (TSPAN-7) 34.2 34.8 258 CD234 Duffy antigen/chemokine
receptor (DARC) 7.7 0.397 259 CD235a Glycophorin-A 55.8 5.11 260
CD243 (BC) 20.8 2.31 261 CD243 (BD) 0.208 0 262 CD244 Natural
killer cell receptor 2B4 0.548 0 263 CD245 99.2 13.3 264 CD249
Glutamyl aminopeptidase (EAP) 19.7 0 265 CD252 Tumor necrosis
factor ligand superfamily 21.4 20.6 member 4 (TNFSF4) 266 CD253
Tumor necrosis factor ligand superfamily 44.1 7.07 member 10
(TNFSF10) 267 CD254 RANKL, TNFSF11 12.3 3.85 268 CD255 10.1 0.437
269 CD256 Tumor necrosis factor ligand superfamily 7.94 0.792
member 13 (TNFSF13) 270 CD257 Tumor necrosis factor ligand
superfamily 63.2 5.03 member 13B (TNFSF13B) 271 CD258 Tumor
necrosis factor ligand superfamily 3.17 0 member 14 (TNFSF14) 272
CD261 Tumor necrosis factor receptor superfamily 30.3 21.4 member
10A (TNFRSF10A) 273 CD262 Tumor necrosis factor receptor
superfamily 12.1 4.55 member 10B (TNFRSF10B) 274 CD263 Tumor
necrosis factor receptor superfamily 1.47 0 member 10C (TNFRSF10C)
275 CD264 Tumor necrosis factor receptor superfamily 44.9 9.09
member 10D (TNFRSF10D) 276 CD267 Tumor necrosis factor receptor
superfamily 91.8 36.6 member 13B (TNFRSF13B) 277 CD268 Tumor
necrosis factor receptor superfamily 64.6 13.5 member 13C/(BAFF-R)
278 CD269 Tumor necrosis factor receptor superfamily 8.51 2.4
member 17 (TNFRSF17) 279 CD270 Tumor necrosis factor receptor
superfamily 31.6 8.79 member 14 280 CD271 Low-affinity nerve growth
factor receptor 1.63 10.4 (NGFR) 281 CD272 B- and T-lymphocyte
attenuator 33.2 12.3 282 CD273 Programmed cell death 1 ligand 2
92.4 51.7 283 CD274 Programmed cell death 1 ligand 1 23.9 1.12 284
CD275 ICOS Ligand 26 0.904 285 CD276 4Ig-B7-H3 100 97.8 286 CD277
Butyrophilin subfamily 3 member A1 1.55 0 287 CD278 Inducible
T-cell costimulator 0.147 0.0836 288 CD279 Programmed cell death
protein 1 5.5 0.492 289 CD281 Toll-like receptor 1 54.7 2.12 290
CD282 Toll-like receptor 2 0.101 0.529 291 CD283 Toll-like receptor
3 68.9 6.92 292 CD284 Toll-like receptor 4 7.94 0.84 293 CD286
Toll-like receptor 6 76.9 11.4 294 CD288 Toll-like receptor 8 85.6
11.2 295 CD289 Toll-like receptor 9 11.3 0.359 296 CD290 Toll-like
receptor 11 45.1 9.5 297 CD292 Bone morphogenetic protein receptor
type-1A 2.39 0.522 298 CD294 Prostaglandin D2 receptor 2 8.81 34.1
299 CD295 Leptin receptor (Lep-R) 49 73.7 300 CD298
Sodium/potassium-transporting ATPase subunit 99.8 98.9 beta-3 301
CD299 C-type lectin domain family 4 member M 29.5 1.07 302 CD300a
CMRF35-like molecule 8 (CLM-8) 1.82 0.222 303 CD300c CMRF35-like
molecule 6 (CML-6) 37.3 3.76 304 CD300e CMRF35-like molecule 2
(CML-2) 38.7 0.697 305 CD301 C-type lectin domain family 10 member
A 3.39 0.626 306 CD303 C-type lectin domain family 4 member C 66.8
3.33 307 CD304 Neuropilin-1 (NRP-1) 65.2 0.502 308 CD305
Leukocyte-associated immunoglobulin-like 4.12 0.972 receptor 1
(LIAR-1) 309 CD307 7.08 0.305 310 CD309 VEGFR2/FLK-1/KDR 34.4 14.2
311 CD312 EGF-like module-containing mucin-like hormone 24.8 12.2
receptor-like 2 312 CD314 NKG2-D type II integral membrane protein
38.5 11.6 313 CD317 Bone Marrow Stromal Antigen 2 48.9 25 314 CD318
CUB domain-containing protein 1 71.7 12.3 315 CD319 SLAM family
member 7 27.8 21.9 316 CD321 Junctional adhesion molecule A (JAM-A)
3.81 5.04 317 CD322 Junctional adhesion molecule B (JAM-B/2) 4.37
0.248 318 CD324 Cadherin-1 17.2 0.387 319 CD325
Cadherin-2/N-cadherin 3.83 0.501 320 CD326 Epithelial cell adhesion
molecule (EPCAM) 18.1 0.463 321 CD328 Siglec-7 32 1.99 322 CD332
FGFR2 0.814 0.181 323 CD333 FGFR3 7.78 1.01 324 CD334 FGFR4 1.35
1.76 325 CD335 NCR1 0.669 0.274 326 CD336 NCR2 0.544 0.212 327
CD337 87.3 26.4 328 CD338 ATP-binding cassette sub-family G member
2 49 19.5 329 CD339 Protein jagged-1 1.76 1.22 330 CD340 Receptor
tyrosine-protein kinase erbB-2 94.9 41 331 CD344 Frizzled 4 65.5
17.5 332 CD349 Frizzled 9 87.6 80.3 333 CD351 FCAMR 76.4 28.1 334
CD352 SLAM-6 0.518 0.394 335 CD354 TREM-1 13.6 1.66 336 CD355
Cytotoxic and regulatory T-cell molecule 10.4 1.24 337 CD357 Tumor
necrosis factor receptor superfamily 10.4 1.95 member 18 338
CD358/DR6 45.1 7.63 339 CD360 (BD) 24.9 3.53 340 CD360 (BL) 33 4.5
341 CD362 Syndecan-2 14.7 0.774 342 CD363 Sphingosine 1-phosphate
receptor 1 18.7 0.757 343 CLA 0.277 9.23 344 CLIP 0.138 0 345 DCIR
0.264 0.15 346 EGF-R 33.3 2.02 347 FMC7 0.0776 0 348 HLA-ABC 99.9
99.8 349 HLA-A2 3.52 20.9 350 HLA-DM 0.172 0.14 351 HLA-DR 0.247
0.481 352 HPC 2.14 6.31 353 ITGB7 0.34 0.159 354 LTBR Tumor
necrosis factor receptor superfamily 34.5 87.6 member 3 355 MIC A/B
97.1 0.328 356 Notch1 20.5 4.01 357 Notch2 95.8 22.8 358 Notch3
5.37 2.15 359 PAC-1 0.137 0.971 360 Podoplanin 8.81 2.91 361 SSEA-3
20.7 0.395 362 SSEA-4 87.4 2.44 363 Stro-1 18.5 6.27 364 TCR alpha
0.327 0.195 beta 365 TCR gamma 52.9 11.1 delta 366 TPBG 0.197 0.178
367 VB8 TCR 25.1 3.93 368 VD2 TCR 13.2 12.1 369 fMLP-R 11.4
0.641
Example 3--Luminex Assay
[0218] A luminex assay was used to quantitate different cytokines
in the conditioned media from Lonza cells and IMP cell cultures.
Data is shown in pg/.mu.g of RNA, this is to standardise the data
relevant to the number of cells in culture.
TABLE-US-00002 pg/ug RNA Cytokine/Chemokine MSCs IMPs Result IL-6
162.4 596 Increase IL-8 6.9 59.8 Increase IP-10 1.4 13.7 Increase
MCP-1 75.8 322.5 Increase RANTES 1.07 125.3 Increase IL-10 0.8 0.1
Decrease IL-12 (p70) 41.6 21.9 Decrease
* * * * *