U.S. patent application number 14/430875 was filed with the patent office on 2015-08-27 for method for polyclonal stimulation of t cells by mobile nanomatrices.
The applicant listed for this patent is MILTENYI BIOTEC GMBH. Invention is credited to Mario Assenmacher, Alexander Scheffold.
Application Number | 20150240204 14/430875 |
Document ID | / |
Family ID | 46940385 |
Filed Date | 2015-08-27 |
United States Patent
Application |
20150240204 |
Kind Code |
A1 |
Scheffold; Alexander ; et
al. |
August 27, 2015 |
METHOD FOR POLYCLONAL STIMULATION OF T CELLS BY MOBILE
NANOMATRICES
Abstract
The present invention provides a method for polyclonal
stimulation of T cells, the method comprising contacting a
population of T cells with a nanomatrix, the nanomatrix comprising
a) a matrix of mobile polymer chains, and b) attached to said
matrix of mobile polymer chains one or more stimulatory agents
which provide activation signals to the T cells; thereby activating
and inducing the T cells to proliferate; wherein the nanomatrix is
1 to 500 nm in size. At least one first and one second stimulatory
agents are attached to the same or to separate mobile matrices. If
the stimulatory agents are attached to separate nanomatrices,
fine-tuning of nanomatrices for the stimulation of the T cells is
possible. Closed cell culture systems also benefit from this
method.
Inventors: |
Scheffold; Alexander;
(Berlin, DE) ; Assenmacher; Mario;
(Bergisch-Gladbach, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
MILTENYI BIOTEC GMBH |
Bergisch Gladbach |
|
DE |
|
|
Family ID: |
46940385 |
Appl. No.: |
14/430875 |
Filed: |
September 25, 2013 |
PCT Filed: |
September 25, 2013 |
PCT NO: |
PCT/EP2013/069854 |
371 Date: |
March 24, 2015 |
Current U.S.
Class: |
424/93.71 ;
435/375 |
Current CPC
Class: |
C12N 5/0075 20130101;
C12N 2539/00 20130101; C12N 2501/515 20130101; C12N 2501/51
20130101; A61P 37/04 20180101; C12N 5/0636 20130101; C12N 5/0637
20130101; C12N 2501/998 20130101; A61K 35/17 20130101; C12N 2533/70
20130101 |
International
Class: |
C12N 5/0783 20060101
C12N005/0783; A61K 35/17 20060101 A61K035/17 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 25, 2012 |
EP |
12185939.1 |
Claims
1. The use of a nanomatrix for in-vitro polyclonal stimulation of T
cells, the nanomatrix comprising a) a matrix of mobile polymer
chains, and b) attached to said matrix of mobile polymer chains one
or more stimulatory agents which provide activation signals to the
T cells; wherein the nanomatrix is 1 to 500 nm in size.
2. An in vitro method for polyclonal stimulation of T cells, the
method comprising contacting a population of T cells with a
nanomatrix, the nanomatrix comprising a) a matrix of mobile polymer
chains, and b) attached to said matrix of mobile polymer chains one
or more stimulatory agents which provide activation signals to the
T cells; thereby activating and inducing the T cells to
proliferate; wherein the nanomatrix is 1 to 500 nm in size.
3. The method according to claim 2, wherein at least one first and
one second stimulatory agents are attached to the same matrix of
mobile polymer chains.
4. The method according to claim 2, wherein at least one first and
one second stimulatory agents are attached to separate matrices of
mobile polymer chains.
5. The method according to claim 4, wherein the ratio of
nanomatrices to cells is larger than 500:1 allowing fine-tuning of
T cell stimulation.
6. The method according to any one of claims 2 to 5, wherein one
stimulatory agent is an anti-CD3 antibody or fragment thereof.
7. The method according to any one of claims 1 to 6, wherein the
second stimulatory agent is an anti-CD28 antibody.
8. The method according to any one of claims 2 to 7, wherein the
matrix of mobile polymer chains consists of a polymer of
dextran.
9. The method according to any one of claims 2 to 8, wherein the
nanomatrix carries magnetic, paramagnetic or superparamagnetic
nano-crystals, embedded into the matrix of mobile polymer
chains.
10. The method according to any one of claims 2 to 9, wherein the
stimulatory agent is attached at high density with more than 25
.mu.g per mg nanomatrix.
11. The method according to any one of claims 2 to 10, wherein the
stimulated T cells are Treg cells.
12. A method for polyclonal stimulation of T cells in a closed cell
culture system, the method comprising contacting a cell culture
comprising a population of T cells within said closed cell culture
system with a dosage of nanomatrix, the nanomatrix comprising a) a
matrix of mobile polymer chains; and b) attached to said matrix of
mobile polymer chains one or more stimulatory agents which provide
activation signals to the T cells; wherein the nanomatrix is 1 to
500 nm in size, and wherein said dosage of nanomatrix is applied
sterile to said closed cell culture system, and wherein said dosage
depends on the volume of the cell culture in said closed cell
culture system.
13. A method according to claim 12, wherein said volume of the cell
culture can be determined by a balance or a camera system without
affecting the sterility barrier.
14. A method according to claims 12 and 13, wherein determining and
applying said dosage are performed automatically.
15. A pharmaceutical composition comprising a population of
stimulated T cells produced according to the method of claims
12-14.
Description
FIELD OF INVENTION
[0001] The present invention relates generally to the field of
immunology, in particular to processes for polyclonal stimulation
of T cells by nanomatrices.
BACKGROUND OF THE INVENTION
[0002] Antibodies against CD3 are a central element in many T cell
proliferation protocols. Immobilized on a surface, anti-CD3
delivers an activating and proliferation-inducing signal by
crosslinking of the T cell receptor complex on the surface of T
cells. By immobilizing anti-CD3 and anti-CD28 to simultaneously
deliver a signal and a co-stimulatory signal, proliferation can be
increased (Baroja et al (1989), Cellular Immunology, 120: 205-217).
In W009429436A1 solid phase surfaces such as culture dishes and
beads are used to immobilize the anti-CD3 and anti-CD28 antibodies.
Regularly, the immobilization on beads is performed on
DynaBeads.RTM.M-450 having a size of 4.5 .mu.m in diameter.
[0003] EP01257632B1 describes a method for stimulating a population
of T-cells by simultaneous T-cell concentration and cell surface
moiety ligation that comprises providing a population of cells
wherein at least a portion thereof comprises T-cells, contacting
the population of cells with a surface, wherein the surface has
attached thereto one or more agents that ligate a cell surface
moiety of at least a portion of the T-cell and stimulates at least
that portion of T cells or a subpopulation thereof and applying a
force that predominantly drives T-cell concentration and T-cell
surface moiety ligation, thereby inducing T-cell stimulation. The
term force as used herein refers to a force used to drive the cells
and may include a variety of forces that function similarly, and
include a force greater than gravitational force, a hydraulic
force, a filtration force generated by transmembrane pressure, a
centrifugal force, or a magnetic force. EP1257632B1 describes that
ratios of particles to cells can vary, however certain preferred
values include at least 1:4 to 6:1, with one preferred ratio being
at least 2:1 beads per T-cell. Regularly, DynaBeads.RTM.M-450
having a size of 4.5 .mu.m in diameter coupled to anti-CD3 and
anti-CD28 antibodies were used in experiments in a bead/T-cell
ratio of 3:1. Again, these methods use solid phase surfaces to
co-immobilize T cell stimulation agents such as anti-CD3 and
anti-CD28 antibodies. These surfaces are cell-sized and comparable
with the T cells themselves.
[0004] US2008/0317724A1 discloses that the spatial presentation of
signal molecules can dramatically affect the response of T cells to
those signal molecules. For example, when anti-CD3 and anti-CD28
antibodies are placed on separate predefined regions of a
substrate, T cells incubated on the substrate secrete different
amounts of interleukin-2 and/or exhibit spikes in calcium,
depending not only on the types but also on the spacing of these
signal molecules. For example, a pattern was generated with
anti-CD3 and anti-CD28 antibodies, where anti-CD3 antibodies
occupied a central feature surrounded by satellite features of
anti-CD28 antibodies that were spaced about 1 to 2 microns from the
central anti-CD3 feature. When the anti-CD28 antibody features were
spaced about 1 to 2 microns apart, the T cell secretion of
interleukin-2 (IL-2) was enhanced compared to when the anti-CD3 and
anti-CD28 antibodies were presented together to the T cells in
"co-localized" features.
[0005] The publication of Erin R Steenblock and Tarek M Fahmy
(Molecular Therapy vol. 16 no. 4, 765-772 April 2008) uses
solid-surface nanoparticles (130 nm) and show that these
nanoparticle stimulate T cells weaker than microparticles (8
.mu.m). The authors stated that these findings are supported by
those of previous reports (Mescher, MF (1992). J Immunol 149:
2402-2405.), demonstrating that micron-sized particles, which are
close in size to T cells, provide optimal T-cell stimulation.
Mesher's study demonstrated the critical importance of a large,
continuous surface contact area for effective CTL activation. Using
class I alloantigen immobilized on latex microspheres, particle
sizes of 4 to 5 microns were found to provide an optimum stimulus.
Below 4 microns, responses decreased rapidly with decreasing
particle size, and large numbers of small particles could not
compensate for suboptimal size.
[0006] U.S. Pat. No. 8,012,750B2 discloses a biodegradable device
for activating T-cells. The biodegradable support is first
formulated into a shape, such as a microsphere. The biodegradable
supports then coated with a first material providing a reactive
surface which is capable of binding to second materials. The second
materials have a reactive surface which permits binding to surface
structures on a cell. The biodegradable support can be formulated
into various shapes. Microspheres are a preferred formulation
because of the simplicity of manufacture and the spherical shape
allows an increased surface area for interaction with cellular
receptors. According to U.S. Pat. No. 8,012,750B2 nanospheres do
not provide enough cross-linking to activate naive T-cells and thus
can only be used with previously activated T-cells. Again,
experimental data were generated with spheres co-immobilized with
anti-CD3 and anti-CD28 antibodies ranging in size from 4 to 24
microns with a mean of 7 microns.
[0007] US2012/121649A1 discloses methods of expanding
antigen-specific anti-tumorigenic T cells comprising administering
to a subject or to cells in vitro an antigen/MHC/co-stimulatory
molecule/nanoparticle complex in an amount sufficient to stimulate
expansion of an antigen-specific anti-tumorigenic T cell. The
nanoparticle is from about 1 nm to 10 nm. The abilities of the pMHC
and pMHC/anti-CD28 mAb-conjugated nanoparticles to stimulate and
activate cognate naive CD8+ T cells were compared. The nanoparticle
can further comprise one or more of a biodegradable coating formed
from dextran and other molecules. E.g. iron (III) oxide and PLGA
can be combined to form nanoparticle resulting in a solid surface
particle. US2012/121649A1 does not disclose a polyclonal
stimulation of T cells, e.g. with anti CD3 and antiCD28 mAbs
immobilized on nanoparticles.
[0008] US2010/284965A1 discloses methods for activating T cells by
administering to the cells polymeric nano- or microparticles which
have an anti-CD28 antibody attached. The microparticles are in the
range of between 0.5 and 1000 microns and nanoparticles in the
range of between 50 nm to less than 0.5 nm. The particles consist
of PLGA and thus represent spherically shaped solid surface
particles. In addition, US2010/284965A1 does not disclose
polyclonal stimulation with the nanoparticles.
[0009] Taken together, beads or microspheres used in the state of
the art for polyclonal T cell activation via immobilized T cell
stimulatory antibodies regularly are cell-sized (mostly 1 to 10
.mu.m in size), uniformly round-shaped particles. Beads of this
size have several disadvantages with regard to their potential to
interact with T cells as well as their production, handling and
safety in clinical T cell therapy procedures. [0010] 1. Due to the
solid surface of the bead the size of interaction area between the
bead and cells is limited. [0011] 2. Their preparation is complex
and costly as compared to soluble antibodies and it is especially
inconvenient to generate them in cGMP quality, e.g. due to their
size no sterile filtration is possible, sedimentation complicates
handling, i.e. constant particle number/volume during filling and
antibody loading. [0012] 3. They are inconvenient to use for in
vitro processes to generate T cell therapeutics for in vivo use,
[0013] since they have to be added to cells in defined cell/bead
ratios at defined density cell/beads per surface area, [0014]
adaption of stimulation strength is only possible to some extent,
since the T cell stimulation strength is mostly determined by the
density of antibodies on the cell surface and not by the number of
beads/cell [0015] aliquoting is inaccurate due to sedimentation,
[0016] sterile filtration is not possible [0017] due to their size
they might affect cell viability and function and they cannot
simply be removed from cells by centrifugation. Therefore either
special protocols for "bead removal" or biodegradable particles
have been developed. However both methods suffer from inaccuracies
with regard to the actual number of residual beads after the
removal process, leaving behind a certain risk for toxic effects if
T cell stimulatory beads are injected into patients. This problem
is particularly relevant because of the size of the particles,
since each single particle on its own might still have retained the
capacity to activate T cells in vivo.
[0018] Therefore, there is a need for an improved in-vitro method
for polyclonal T cell stimulation.
SUMMARY OF THE INVENTION
[0019] Surprisingly, it was found that polyclonal T cell
stimulatory agents such as antibodies, e.g. against CD3 and CD28,
attached to nanomatrices, which are characterised by a mobile
polymeric matrix backbone (non-solid surface), which may have
embedded within the matrix additional functional compounds, such as
magnetic nanocrystals, can be used to stimulate naive and memory T
cells in vitro, although their diameter is smaller than 1 .mu.m,
preferentially smaller than 500 nm, more preferentially smaller
than 200 nm. Contrary thereto, it was found that beads with solid
surfaces of the same size as the nanomatrices used herein are not
able to stimulate T cells at all or to a similar level like the
nanomatrices which is in accordance with the well established
opinion of the person skilled in the art. Due to their small size
the nanomatrices per se, without antibodies attached thereto, do
not alter structure, function, activity status or viability of
cells, i.e. they do not cause perturbance in the cells and do not
interfere with subsequent analyses, experiments and therapeutic
applications of the stimulated cells. In addition, preferentially,
the nanomatrix is biodegradable and non-toxic to living cells, i.e.
the nanomatrix is a biologically inert entity with regard to
alterations of the cell function. Therefore the nanomatrix used in
the method of the present invention improves the in-vitro
stimulation of T-cells by saving the viability of the cells. In
addition sterile filtration of the small nanomatrices is possible
which is an important feature for long term T cell in vitro
expansion under conditions which are compliant with rigorous GMP
standards and is a valuable option for clinical application of the
in vitro expanded T cells. Moreover these nanomatrices are well
suited for use in sterile and closed cell culture system such as
described e.g. in WO2009/072003 (CliniMACS.RTM.Prodigy, Miltenyi
Biotec GmbH, Germany).
[0020] In addition surprisingly, it was found that the T cell
stimulatory agents such as antibodies, e.g. against CD3 and CD28,
attached to nanomatrices may be conjugated to separate nanomatrices
(instead of conjugating to the same nanomatrix), which can be mixed
hereafter for optimised use. In general, the ratio of nanomatrices
to cells is larger than 100:1, preferentially larger than 500:1,
most preferentially larger than 1000:1. This results in the
possibility of fine-tuning of the nanomatrices used for stimulation
of the target T cells, e.g. it facilitates the production process
and quality control of the single nanomatrices and improves the
flexibility of the reagent, e.g. facilitating the optimisation of
the activation conditions for specialised T cell subsets by
titrating various CD3 and CD28 concentrations and ratios.
[0021] In a first aspect the present invention provides the use of
the nanomatrices disclosed herein for the polyclonal in-vitro
stimulation of T cells.
[0022] In a further aspect the present invention provides a method
for polyclonal stimulation of T cells, the method comprising
contacting a population of T cells with a nanomatrix, wherein the
nanomatrix comprises a matrix of mobile polymer chains, and has
attached thereto one or more agents which provide activation
signals to the T cells; thereby activating and inducing the T cells
to proliferate; and wherein the nanomatrix is 1 to 500 nm,
preferentially 10 to 200 nm, in size. Preferentially, the
nanomatrix is biologically inert with regard to alterations of the
cell function. In addition preferentially, the nanomatrix is
biodegradable.
[0023] The stimulated and optionally expanded T cells achieved with
the present invention can be used in subsequent therapeutic or
non-therapeutic applications without the need for eliminating or
removing the nanomatrix due to the property of the nanomatrix of
being biologically inert with regard to alterations of the cell
function
[0024] Alternatively, due to being soluble or colloidal the
nanomatrices can easily be diluted by repeated washing steps to
effective concentrations below the T cell activation threshold
after the T-cell stimulation process.
[0025] The nanomatrix is 1 to 500 nm, preferentially 10 to 200 nm
in size. The nanomatrix is a mobile matrix consisting of a
polymeric material but has no solid phase surface in contrast to
beads or microspheres. Agents such as anti-CD3 and/or anti-CD28
antibodies which allow for polyclonal stimulation of T cells are
attached to the mobile polymer chains of the matrix. Within the
matrix additional substances, such as magnetic nanocrystals,
fluorescent dyes, etc., can be embedded and add additional
functions to the nanomatrix without altering its basic mobile
structure, surface features, or cell interaction parameters of the
nanomatrix.
[0026] This mobility, i.e. flexibility of polymeric matrices in
aqueous solutions even when attached to solid surfaces is well
known in the field. For example, a quantitative estimation of the
flexibility or mobility of dextran and other polymers used for
biological or medical applications is given in Bertholon et al.
Langmuir 2006, pp 45485-5490.
[0027] The mobility introduced to the stimulation matrix by the
used polymer is well described in multiple publications for
polysaccharides and embedded superparamagnetic nanocrystals, which
are frequently used for in vivo imaging technologies in clinical
situations. These reagents are very similar to the type of reagent
which was used here in several of the examples given. It is well
known that despite the term "particle" these reagents are actually
not comparable to spherical solid surface particles:
"Most of the colloidal (so called) nanoparticles used for medical
purpose are coated with polymer or polyelectrolytes and they cannot
be really considered as rigid spheres (Di Marco et al. Int J.
Nanomed. 2007, p618, line 5 ff.)".
[0028] To exemplify directly: this typical feature of
polysaccharide embedded superparamagnetic nanocrystals is best
described by the discrepancy in the size values obtained by
different methods used for size determination. Typically
transmission electron microscopy uses dried samples and thus
determines mainly the size of the embedded nanocyrstals, which are
always in the range of 10 nm. However, in contrast dynamic laser
scattering which also takes into consideration the size of the
surrounding matrix in aqueous solution, determines much larger
diameters (e.g. 5-10 nm versus 80-150 nm for AMI-25 a clinical
contrast reagent, consisting of a dextran matrix and embedded
ironoxide crystals. This means that >99% of the total volume of
the complex in aqueous solution is made up by the mobile matrix
(Wang et al. Eur. Radiol. 2001: page 2323). For the matrices which
were used for example in Example 1, size measurement revealed 30 nm
(TEM) and 65 nm (DLS), thus about 80-90% of the complex in aqueous
solution consists of the mobile matrix. Therefore in these types of
mobile matrices, the mobile polymer determines the biophysical
behaviour during interaction with cells, rather than a stiff
particle or solid surface covered, which might in addition be
coated with a thin layer of polymer used for attachment. Thus the
well-known flexibility or mobility (motility) of the polymer chains
in the described matrix is the determining feature and critical
difference to solid surface attached antibodies on microspheres
classically used for stimulation.
[0029] In summary, these examples indicate the current common
knowledge about the behaviour of mobile, flexible polymer matrices
in aqueous solution (prerequisite for all cell culture
applications) and highlight the fact that also the biophysical
properties of polymer coated colloids, such as the ironoxide
containing polysaccharide matrices used in this study but not
limited to those, are determined mainly by the feature of polymer
material, independent of the embedded substances.
[0030] Therefore the present invention provides an in vitro method
for polyclonal stimulation of T cells, the method comprising
contacting a population of T cells with a nanomatrix, the
nanomatrix comprising
a) a matrix of mobile polymer chains; and b) attached to said
matrix of mobile polymer chains one or more stimulatory agents
which provide activation signals to the T cells; thereby activating
and inducing the T cells to proliferate, wherein the nanomatrix is
1 to 500 nm, preferentially 10 to 200 nm, in size, and wherein the
majority (i.e. more than 50%), preferentially more than 80% and
more preferentially more than 90% and most preferentially more than
99% of the total volume of the nanomatrix in aqueous solution
consists of mobile polymer chains.
[0031] If additional substances, such as magnetic nano-crystals,
fluorescent dyes, etc. are embedded into the matrix, the resulting
complex consists mainly of mobile polymer chains, making up more
than 50%, preferentially more than 80% and more preferentially more
than 90% and most preferentially more than 99% of the volume in
aqueous solution.
BRIEF DESCRIPTION OF THE DRAWINGS
[0032] FIG. 1: CD3/CD28 Nanomatrices for naive T cell
expansion.
[0033] Sorted human naive CD4 and CD8 T cells were stimulated with
CD3/CD28 conjugated nanomatrices at the indicated concentrations
(effective CD3 concentration) in the presence of IL-2 for 7 days.
Nanomatrices conjugated with CD3 and CD28 in various indicated
ratios were compared. As a high control CD3/CD28 conjugated
MACSiBeads were used. The absolute number of viable cells in the
culture at day 7 is given.
[0034] FIG. 2 A-C: Nano-sized solid particles versus Nanomatrix for
stimulation of T cells. Nano-sized solid particles (AdemtechBeads,
diameter=200 nm) were conjugated with CD3/CD28 (1:1) and used for
stimulation of naive CD4 and CD8 T cells at the indicated
concentrations of CD3. As activation parameters either CD3
down-regulation (FIG. 2A, day 3) or induction of the early
activation markers CD25 (day 3 FIG. 2B, day 5 FIG. 2C) were
analysed by flow-cytometry. The values were normalized to the value
of the same cells stimulated via CD3/CD28 nanomatrix (100 ng/ml
CD3). The values from 4 different donors are given.
[0035] FIG. 3: Expansion of sorted human T cell populations: Human
T cells were sorted into various subpopulations (total T cells,
total naive T cells, naive CD4+ T cells, naive CD8+ T cells) and
stimulated with CD3/CD28 conjugated nanomatrices at the indicated
concentrations (effective CD3 concentration) and a CD3/CD28 ratio
of 1:1 in the presence of IL-2 for 7 days. As a high control
CD3/CD28 conjugated MACSiBeads were used. The absolute number of
viable cells in the culture at day 7 is given.
[0036] FIG. 4: Expansion of human Treg: CD25+ Treg were isolated
from PBMC by magnetic CD25 selection and expanded in the presence
or absence of 100 nM Rapamycin for 14 days using CD3/CD28
nanomatrix (200 ng/ml CD3) and high dose IL-2. On day 7 the cells
were restimulated by adding fresh nanomatrix+IL-2. On day 14 the
number of viable Treg was determined. The frequency of Foxp3
expressing cells was determined by intracellular
immunofluorescence. Each dot represents an individual healthy
donor.
[0037] FIG. 5: Comparison of CD3 and CD28 conjugated to different
nanomatrices versus conjugated to the same nanomatrix. Sorted human
naive CD4 and CD8 T cells were stimulated either with CD3/CD28
conjugated nanomatrices or CD3 and CD28 conjugated to different
nanomatrices at the indicated concentrations (effective CD3
concentration) in the presence of IL-2 for 7 days. As a high
control CD3/CD28 conjugated MACSiBeads were used. The absolute
number of viable cells in the culture at day 7 is given (A).
Results from two donors are depicted. In addition the cells were
labelled with CFSE and the proliferative activity measured on day 5
after activation (1 representative donor) (B).
[0038] FIG. 6: Comparison of soluble CD3 or CD28 antibodies to CD3
or CD28 antibodies conjugated to nanomatrices. Naive CD4 and CD8 T
cells were isolated and stimulated in vitro in the presence of IL-2
using soluble CD3 (0-10000 ng/mL) w/o CD28 (A) or in the presence
of soluble CD28 (200 ng/ml) (B) and compared to CD28 conjugated to
nanomatrix (200 ng/ml) and analysed for early activation markers
CD25/CD69 on day 5 or for expansion (C) on day 7. 2 donors were
analysed in duplicates.
[0039] FIG. 7: Transduction efficiency of isolated T cell subsets
stimulated with various stimulation agents. T cell subsets, naive
(T.sub.N, CD62L+CD45RA+), central memory (T.sub.CM, CD62L+CD45RA-)
and effector (T.sub.EM, CD62L-CD45RA-) T cells activated using
CD3/CD28 nanomatrices, plate-bound CD3+soluble CD28 or CD3/CD28
conjugated MACSibeads and transduced them using a retroviral vector
expressing a TCR specific for MART-1. As a standard total PBMC were
activated using soluble CD3/CD28. The frequency of transduced cells
expressing the MART-1 TCR was determined using a fluorescently
labelled MART-1/HLA-A2 tetramer.
[0040] FIG. 8: Enriched T cell subsets expand at least like PBMC or
better.
[0041] CD8.sup.+ T cell subsets from freshly isolated PBMC from
melanoma patients were isolated and the stimulated with coated CD3
plus soluble CD28 (CD3+CD28 in the graph) or with CD3/CD28/CD2
coated MACSiBeads (MACSiBeads in the graph) or with CD3/CD28
Nanomatrix in the presence of IL2. After 2 days of stimulation
cells have been transduced to express MART-1 TCR. In the graph is
reported the fold expansion of each culture at day 13-15 after
stimulation. The fold expansion values are relative to soluble CD3
stimulated PBMC that shows a fold expansion of 57.61.+-.17.75
[0042] FIG. 9: Naive and central memory cells stimulated with
MACSiBeads or CD3/CD28 nanomatrix show a less terminal
differentiated phenotype. PBMC were freshly isolated from
leukapheresis of melanoma patients. CD8+ T cell subsets were
enriched and then stimulated with CD3+CD28 or MACSiBeads or
CD3/CD28 nanomatrix in the presence of IL2. PBMC instead were
stimulated with soluble CD3 and IL2. Cells were transduced to
express MART-1 TCR 48 h after stimulation. The data here were
obtained 13-15 days after stimulation of each culture. Frequencies
of A) MART-1 tetramer.sup.+ CD62L.sup.+ and B) MART-1
tetramer.sup.+CCR7.sup.+cells among CD8.sup.+ T cells are shown.
After IL2 withdrawal cells were stained for CD27, CD57 markers or
stimulated with a MART-1.sup.+ HLA-A2.sup.+ melanoma cell line in
the presence of CD107a antibody and Monensin for 5 h. Statistical
analysis of C) MART-1 tetramer.sup.+ CD27.sup.+; D) MART-1
tetramer.sup.+ CD57.sup.+ and E) CD107a.sup.+ cells among CD8.sup.+
T cells frequencies.
[0043] FIG. 10: Cytokine secretion upon MART-1 restimulation in
CD8.sup.+ T cell subsets. CD8.sup.+ T cell subsets from freshly
isolated PBMC from melanoma patients were isolated and the
stimulated with CD3+CD28 MACSiBeads or with CD3/CD28 nanomatrix in
the presence of IL2. PBMC from the same melanoma patient were
stimulated with soluble CD3 and IL2. After 2 days of stimulation
cells have been transduced to express MART-1 TCR and cultured for a
total 13-15 days. Afterwards cells were washed out from IL2 and
rested for further 2 days and then restimulated with MART-1.sup.+
HLA-A2.sup.+ melanoma cell line for 6 h. The cytokine production
was determined by intracellular staining. Graphs show the
frequencies of A) IFN.gamma..sup.+; B) IL2.sup.+ and C)
TNF.alpha..sup.+ CD8+ T cells.
DETAILED DESCRIPTION OF THE INVENTION
[0044] It was a well established opinion in the scientific
community that particles smaller than 1 .mu.m are not convenient to
stimulate T cells effectively because such small particles do not
provide enough cross-linking to activate T cells. Therefore,
generally, beads or microspheres with solid phase surfaces used to
stimulate polyclonally T cells are always larger than 1 .mu.m in
size in the state of the art, regularly they are cell-sized.
[0045] Now unexpectedly, the inventors found that nanomatrices
being smaller than 1 .mu.m, preferentially smaller than 500 nm,
more preferentially smaller than 200 nm, having a mobile matrix and
having attached thereto polyclonal stimulatory agent(s) are
convenient to stimulate T cells. It is essential to the present
invention that the nanomatrix being smaller than 500 nm has no
solid phase surface (resulting in a flexible and mobile phase) in
contrast to beads or microspheres of the same size (see Example 3).
The nanomatrix is like a mesh or net consisting of a mobile
polymeric material, preferentially dextran. The nanomatrix is very
plastic resulting in the ability to snuggle to the cell surface
membrane of target cells, i.e. the T cells which shall be
activated. Therefore, the nanomatrix binds with its agents attached
to the mobile matrix to the respective receptors (antigens) on the
cell surface, whereby the flexibility of the matrix allows optimal
interaction with the binding partners. To a certain degree the
shape of the nanomatrix adapts to the target cell surface thereby
extending the contacting surface between nanomatrix and target
cell. Due to the size of the nanomatrix of 1 to 500 nm,
preferentially 10 to 200 nm, they are too small to cause
perturbance in the cell, i.e. the nanomatrix is biologically inert
with regard to alterations of the cell function. Such perturbances
triggered by direct cell/bead contact is problematic if beads or
microspheres of 1 .mu.m or larger in size are used. In addition,
preferentially, the nanomatrix is biodegradable and non-toxic to
the cells due to the composition consisting of biodegradable
polymeric material such as a polymer of dextran. In consequence,
the nanomatrix is a completely biologically inert entity with
regard to alterations of the cell function but biodegradable.
Therefore there is no need to remove the nanomatrix after
contacting it with the T cells for stimulation and proliferation.
No disturbing effects occur due to the presence of the nanomatrices
in an activated T cell composition for subsequent analysis,
experiments and/or clinical applications of these cells.
[0046] In addition, due to being soluble or colloidal the unbound
nanomatrices can easily be diluted by repeated washing steps to
effective concentrations below the T cell activation threshold
after the T-cell stimulation process.
[0047] The mobile matrix of the nanomatrix has attached thereto one
or more stimulatory agents which provide activation signal(s) to
the T cells, thereby activating and inducing the T cells to
proliferate. The agents are molecules which are capable of binding
to a cell surface structure and induce the polyclonal stimulation
of the T cells. One example for agents attached to the mobile
matrix of the nanomatrix is anti-CD3 monoclonal antibody (mAb) in
combination with a co-stimulatory protein such as anti-CD28 mAb.
Other examples are anti-CD2, anti-CD137, anti-CD134, Notch-ligands,
e.g. Delta-like1/4, Jagged1/2 either alone or in various
combinations with anti-CD3. T cells to be stimulated are e.g. naive
T cells, memory T cells, CD4 Treg and CD8 Treg cells.
Preferentially, the stimulatory agent attached to the mobile matrix
of the nanomatrix is anti-CD3 monoclonal antibody (mAb) in
combination with the co-stimulatory protein anti-CD28 mAb.
[0048] Therefore, in one aspect the present invention provides a
method for polyclonal stimulation of T cells, the method comprising
contacting a population of T cells with a nanomatrix, the
nanomatrix comprising
a) a matrix of mobile polymer chains; and b) attached to said
matrix of polymer chains one or more stimulatory agents which
provide activation signals to the T cells; thereby activating and
inducing the T cells to proliferate; and wherein the nanomatrix is
1 to 500 nm, preferentially 10 to 200 nm, in size.
[0049] The nanomatrix may be biologically inert with regard to
alteration of the cell function.
[0050] In addition, or alternatively, the nanomatrix may be
biodegradable.
[0051] The nanomatrix may be of cGMP quality for clinical
applications. Sterility can be achieved e.g. by sterile filtration
using filters with suitable pore size (200 nm) or by other methods
well known by the person skilled in the art. GMP processing of cell
products is frequently performed in closed cell culture systems.
Use of the nanomatrix in GMP cultivation systems is of advantage
due to: [0052] feature of nanomatrix to pass a sterile filter
[0053] a low particle density or colloidal behaviour that avoids
faster sedimentation of matrix compared to T cells [0054] dosing of
the nanomatrix in relation to the volume of the culture rather than
the T cell number.
[0055] The feature of the nanomatrix of being able to pass sterile
filters allows the addition to closed cell culture systems being
equipped or equipable with sterile filters, e.g. cell cultivation
bags (Miltenyi Biotec, Baxter, CellGenics), G-Rex devices (Wilson
Wolf manufacturing), WAVE Bioreactors (GE Healthcare), Quantum Cell
Expansion System (Terumo BCT), CliniMACS.RTM. Prodigy (Miltenyi
Biotec, see Apel et al. 2013, Chemie Ingenieur Technik 85:103-110).
The nanomatrix can be added to closed cell culture systems using a
syringe to push the nanomatrix through the filter or a pump to pull
the nanomatrix through the filter from a bag or a vial (connected
to a vented vial adapter).
[0056] Other agents used to induce T cell proliferation (cell sized
magnetic beads, cell lines) cannot pass a sterile filter. Use of
those reagents would require to connect agents to the closed cell
culture system using a sterile tubing welder (e.g. Terumo, GE,
Genesis). Not all of those tubing welders are designed for
operation within a GMP clean room. Thus adding an agent to the
closed cell culture system through a sterile filter provides a
significant advantage.
[0057] Rigid cell sized magnetic particles used to induce T cell
proliferation have a higher density than lymphocytes including T
cells (1.07 g/l) and thus sediment faster than the nanomatrix.
Agitated cell culture systems such as the WAVE Bioreactor (GE
Healthcare) avoid complete sedimentation of the cells by movement
of the container used for cultivation. Movement of the container
applies different forces to cells/particles of different size and
density, resulting in a relative movement. Induction of
proliferation is reduced within the WAVE Bioreactor compared to
static culture in cell culture flasks.
[0058] The use of the CD3CD28 MACSiBeads ("ExpAct T reg kit", "T
cell expansion kit", Miltenyi Biotec) utilizing rigid cell sized
magnetic particles has been optimized to be used in a given ratio
of beads per cell (e.g. 1:2), requiring determination of the number
of T cells within the culture container and defined cell density,
i.e. an optimized number of cells and beads per surface area,
enabling optimal interaction of beads and cells, which both
sediment to the bottom of the culture vessel within minutes. For a
clinical application of expanded T cells this results in an
additional sampling step, possibly affected sterility of the
product. Use of the nanomatrix of the present invention allows for
dosing of the T cell proliferation inducing agent based on the
volume of the culture, since the reagent can freely diffuse in the
medium and does not sediment. The volume of the T cell containing
cell suspension can be determined by a balance or a camera system
without affecting the sterility barrier. Dosing of the agent based
on volume determination allows for a completely automated cell
product manufacturing process (e.g. using the CliniMACS.RTM.
Prodigy system) without manual cell counting steps.
[0059] The contacting can occur e.g. in vitro in any container
capable of holding cells, preferably in a sterile environment. Such
containers may be e.g. culture flasks, culture bags, bioreactors or
any device that can be used to grow cells (e.g. the sample
processing system of WO2009072003, i.e. the CliniMACS.RTM. Prodigy
system).
[0060] Therefore, in one aspect the present inventions provides a
method for polyclonal stimulation of T cells in a closed cell
culture system, the method comprising contacting a cell culture
comprising a population of T cells within said closed cell culture
system with a dosage of nanomatrix, the nanomatrix comprising
a) a matrix of mobile polymer chains; and b) attached to said
matrix of mobile polymer chains one or more stimulatory agents
which provide activation signals to the T cells; wherein the
nanomatrix is 1 to 500 nm in size, and wherein said dosage of
nanomatrix is applied sterile to said closed cell culture system,
and wherein said dosage depends on the volume of the cell culture
in said closed cell culture system.
[0061] Said volume of the cell culture can be determined by a
balance or a camera system without affecting the sterility
barrier.
[0062] Determining and applying said dosage may be performed
automatically.
[0063] The nanomatrix used in the present invention can be a
nanomatrix wherein at least one first agent and one second agent
are attached to the same mobile matrix. Nanomatrices of this kind
are contacted with T cells, thereby activating and inducing the T
cells to proliferate. The ratio of the first and the second agent
attached to the same flexible matrix may be in the range of the
ratios of 100:1 to 1:100, preferentially between 10:1 and 1:10,
most preferentially between 2:1 and 1:2.
[0064] In addition surprisingly, it was found that the nanomatrix
of the present invention also can be a nanomatrix wherein at least
one first agent and one second agent are attached to separate
mobile matrices. A mixture of these nanomatrices is contacted with
T cells, thereby activating and inducing the T cells to proliferate
(see Example 6). The ratio and/or concentration of the mobile
matrix having attached thereto the first agent and the mobile
matrix having attached thereto the second agent may vary to yield
optimal stimulation results depending on the kind of T cells used
and/or agents used. This facilitates the optimisation of the
activation conditions for specialised T cell subsets by titrating
various concentrations and ratios of the mobile matrix having
attached thereto the first agent and the mobile matrix having
attached thereto the second agent. It is advantageous that
generally the ratio of nanomatrices to cells is larger than 100:1,
preferentially larger than 500:1, most preferentially larger than
1000:1. The large amount of nanomatrices per cell allows for a
fine-tuning of the separate labelled nanomatrices which would be
impossible with lower ratios of 1:10 to 10:1 commonly used by
stimulation of T cells using cell-sized beads.
[0065] The nanomatrix used in the present invention is a nanomatrix
wherein the mobile matrix consists of a polymeric, preferentially
biodegradable material which is non-toxic to cells. Preferentially,
the nanomatrix used in the present invention is a nanomatrix
wherein the mobile matrix consists of a polymer of dextran. Using
polymeric material such as dextran for generating the matrix
results in a mobile, flexible matrix. The mobility (flexibility or
plasticity) of the matrix is superior compared to more rigid
particles (having a solid phase surface) or beads of the same size,
i.e. nanostructures, for proliferation of cells.
[0066] The nanomatrix used in the present invention can be a
nanomatrix wherein the mobile matrix is the only or at least main
component of the nanomatrix regardless the agents which are
attached thereto.
[0067] But the nanomatrix used in the present invention also can be
a nanomatrix wherein the nanomatrix carries magnetic, paramagnetic,
superparamagnetic nano-crystals, or fluorescent dyes embedded into
the flexible matrix, preferentially embedded into the polymer of
dextran.
[0068] The nanomatrix used in the present invention can be used in
a method for stimulating T-cells with this nanomatrix wherein the
nanomatrix is not removed in subsequent applications of the
stimulated T cells.
[0069] Alternatively, the nanomatrix used in the present invention
also can be used in a method for stimulating T-cells with this
nanomatrix wherein the nanomatrix is removed before subsequent
applications of the stimulated T cells.
[0070] In another aspect the present invention also provides a
composition comprising
i) the nanomatrix, the nanomatrix comprising a) a matrix of mobile
polymer chains, and b) attached to said matrix of polymer chains
one or more agents which provide activation signals to the T cells;
thereby activating and inducing the T cells to proliferate; and
wherein the nanomatrix is 1 to 500 nm, preferentially 10 to 200 nm,
in size ii) a population of T cells which are activated and induced
to proliferate triggered by the contact between said nanomatrix and
cells.
[0071] The nanomatrix may be biologically inert with regard to
alteration of the cell function.
[0072] The nanomatrix may be biodegradable.
[0073] Agents are attached to the same or separate nanomatrices at
high density, with more than 25 .mu.g per mg nanomatrix,
preferentially with more than 50 .mu.g per mg nanomatrix.
[0074] This composition may be convenient for generating T cell
therapeutics for in vivo use (pharmaceutical composition). The
composition also can be used in other subsequent analyses and
experiments.
[0075] In another aspect the present invention also provides a
composition or a pharmaceutical composition comprising a population
of stimulated and (optionally expanded) T cells produced according
to the method of the present invention. The pharmaceutical
composition may comprise a population of stimulated T cells
produced by the method of the present invention, wherein the method
is performed in a closed cell culture system such as the sample
processing system of WO2009072003.
[0076] Nanomatrices can be prepared by various methods known in the
art, including solvent evaporation, phase separation, spray-drying,
or solvent extraction at low temperature. The process selected
should be simple, reproducible and scalable. The resulting
nanomatrices should be free-flowing and not aggregate in order to
produce a uniform syringeable suspension. The nanomatrix should
also be sterile. This can be ensured by e.g. filtration, a terminal
sterilization step and/or through aseptic processing. A preparation
of nanomatrices is described in Example 1.
DEFINITIONS
[0077] The terms "matrix of mobile polymer chains" and "mobile
matrix" as used herein have an interchangeable meaning. The term
"mobile" refers to the common and well described feature of organic
biopolymers such as dextran or others on nanoparticles (see
Bertholon et al. Langmuir 2006, pp 45485-5490). These polymers
consists of mobile (motile), preferentially highly mobile (motile)
chains, so the matrix is characterised by the absence of a solid
surface as the attachment point for the stimulating agents such as
antibodies, and which is in strong contrast to currently used beads
or microspheres which regularly have an inflexible, stiff surface.
As a result the nanomatrix comprising a matrix of mobile polymer
chains is flexible and adjustable to the form of the surface of the
cells. In addition as a result the nanomatrix is a nanomatrix
wherein the majority (i.e. more than 50%), preferentially more than
80% and more preferentially more than 90% and most preferentially
more than 99% of the total volume of the nanomatrix in aqueous
solution consists of mobile polymer chains.
[0078] The contact between nanomatrix which has coupled thereto one
or more stimulatory agents and the cells to be stimulated benefit
from the fact that the nanomatrix has not a fixed, stiff or rigid
surface allowing the nanomatrix to snuggle to the cells. The matrix
consists of a polymeric, preferentially biodegradable or
biocompatible inert material which is non-toxic to cells.
Preferentially the matrix is composed of hydrophilic polymer
chains, which obtain maximal mobility in aqueous solution due to
hydration of the chains. The mobile matrix is the only or at least
main component of the nanomatrix regardless the agents which are
attached thereto.
[0079] The mobile matrix may be of collagen, purified proteins,
purified peptides, polysaccharides, glycosaminoglycans, or
extracellular matrix compositions. A polysaccharide may include for
example, cellulose ethers, starch, gum arabic, agarose, dextran,
chitosan, hyaluronic acid, pectins, xanthan, guar gum or alginate.
Other polymers may include polyesters, polyethers, polyacrylates,
polyacrylamides, polyamines, polyethylene imines, polyquaternium
polymers, polyphosphazenes, polyvinylalcohols, polyvinylacetates,
polyvinylpyrrolidones, block copolymers, or polyurethanes.
Preferentially the mobile matrix is a polymer of dextran.
[0080] The mobile matrix defines the property of the nanomatrix of
being very plastic, i.e. flexible, leading to the ability to
snuggle to the cell surface membrane of target cells, i.e. the T
cells which shall be activated and proliferated. Therefore, the
nanomatrix tightly binds with its agents attached to the mobile
matrix to the cells because the mobility of the matrix provides
optimal access of the attached ligands or antibodies to their cell
surface receptors or antigens. Due to this property the nanomatrix
has the ability to provide enough cross-linking to activate T cells
regardless of the small size of the structure, i.e. smaller than 1
.mu.m, preferentially smaller than 500 nm, more preferentially
smaller than 200 nm, in size. The adaptability of the nanomatrix
caused by the mobile, flexible nanostructure extends the contacting
area between cell surface membrane and the nanomatrix resulting in
more efficient bindings between cell surface molecules and agents
attached to the mobile matrix.
[0081] In some embodiments the mobile matrix may embed magnetic,
paramagnetic or superparamagnetic nano-crystals or other substances
which add additional functional properties such as fluorescent dyes
without altering the basic mobile structure and/or surface
features, i.e. interaction with target cells.
[0082] The term "stimulatory agent" which is attached to the mobile
matrix of the nanomatrix as used herein refers to molecules which
are capable of binding to a cell surface structure and contribute
to a polyclonal stimulation of the T cells. The terms "stimulatory
agent" and "polyclonal stimulatory agent" as used herein have an
interchangeable meaning. Examples of suitable agents for use in the
present invention include agents such as synthesized compounds,
nucleic acids and proteins, including polyclonal or monoclonal
antibodies, and fragments or derivatives thereof, and bioengineered
proteins, such as fusion proteins. In one example, the agents are
mitogenic proteins. Mitogenic proteins are two or more proteins
that are able to deliver the requisite minimum of two signals to
T-cells in order to cause the T-cells to become activated. Examples
of mitogenic proteins are anti-CD3 and anti-CD2 monoclonal
antibodies (mAb) in combination with a co-stimulatory protein such
as and including proteins specific for one or more of the following
T-cell surface molecules: CD28, CD5, CD4, CD8, MHCI, MHCII, CTLA-4,
ICOS, PD-1, OX40, CD27L (CD70), 4-1BBL, CD30L and LIGHT, including
the corresponding ligands to these surface structures, or fragments
thereof. Other suitable agents include agents capable of delivering
a signal to T-cells through cytokine receptors such as IL-2R,
IL-12R, IL-1R, IL-15R; IFN-gammaR, TNF-alphaR, IL-4R, and IL-10R,
including monoclonal antibodies (mAbs) to these receptors, fusion
proteins with a reactive end specific for these receptors and the
corresponding ligands to these receptors or fractions thereof.
Other suitable agents include any agent capable of binding to
cellular adhesion molecules on T-cells such as mAbs, fusion
proteins and the corresponding ligands or fractions thereof to
adhesion molecules in the following categories: cadherins, ICAM,
integrins, and selectins. Examples of adhesion molecules on T-cells
are: CD44, CD31, CD18/CD11a (LFA-1), CD29, CD54 (ICAM-1), CD62L
(L-selectin), and CD29/CD49d (VLA-4). Other suitable agents include
any agents capable of binding to chemokine receptors, including
those in the C-C and C-X-C categories. Examples of chemokine
receptors associated with T-cell function include CCR1, CCR2, CCR3,
CCR4, CCR5, and CXCR3.
[0083] An agent may be attached or coupled to the mobile matrix by
a variety of methods known and available in the art. The attachment
may be covalent or noncovalent, electrostatic, or hydrophobic and
may be accomplished by a variety of attachment means, including for
example, chemical, mechanical, enzymatic, or other means whereby an
agent is capable of stimulating the cells. For example, the
antibody to a cell surface structure first may be attached to the
matrix, or avidin or streptavidin may be attached to the matrix for
binding to a biotinylated agent. The antibody to the cell surface
structure may be attached to the matrix directly or indirectly,
e.g. via an anti-isotype antibody. Another example includes using
protein A or protein G, or other non-specific antibody binding
molecules, attached to matrices to bind an antibody. Alternatively,
the agent may be attached to the matrix by chemical means, such as
cross-linking to the matrix.
[0084] As used herein, the term "antibody" is intended to include
polyclonal and monoclonal antibodies, chimeric antibodies, haptens
and antibody fragments, and molecules which are antibody
equivalents in that they specifically bind to an epitope on the
antigen. The term "antibody" includes polyclonal and monoclonal
antibodies of any isotype (IgA, IgG, IgE, IgD, IgM), or an
antigen-binding portion thereof, including, but not limited to,
F(ab) and Fv fragments such as sc Fv, single chain antibodies,
chimeric antibodies, humanized antibodies, and a Fab expression
library.
[0085] The term "biologically inert" as used herein refers to the
properties of the nanomatrix, that it is non-toxic to living cells
and does not induce strong alterations of the cell function via
physical interaction with the cell surface, due to its small size,
except the specific ligand/receptor triggering function of the
attached ligands or antibodies. The nanomatrices, in addition, may
be biodegradable, e.g. degraded by enzymatic activity or cleared by
phagocytic cells. The biodegradable material can be derived from
natural or synthetic materials that degrade in biological fluids,
e.g. cell culture media and blood. The degradation may occur using
enzymatic means or may occur without enzymatic means. The
biodegradable material degrades within days, weeks or few months,
which may depend on the environmental conditions it is exposed to.
The biodegradable material should be non-toxic and non-antigenic
for living cells and in humans. The degradation products must
produce non-toxic by-products. An important aspect in the context
of being biologically inert is the fact that the nanomatrix does
not induce strong alteration in structure, function, activity
status or viability of labelled cells, i.e. it does not cause
perturbance of the cells and does not interfere with subsequent
experiments and therapeutic applications of the stimulated cells.
The mechanical or chemical irritation of the cell is decreased due
to the properties of the nanomatrix of being very small, i.e.
nano-scale range, and having a mobile matrix which rather snuggles
to the cell surface than altering the shape of the cell surface or
exerting strong shearing force to the cells, e.g. resulting in
membrane rupture.
[0086] The term "PBMC" as used herein refers to peripheral blood
mononuclear cells isolated from human peripheral blood preparations
e.g. by use of a density gradient (e.g. Ficoll, PanColl). "PBMC"
consist of lymphocytes and monocytes.
[0087] The term "purified T cells" as used herein refers to cell
populations with an increased content of T cells. T cells may be
purified by different methods, including FACS.RTM. sorting,
enrichment by magnetic beads (e.g. CD4 and CD8 MicroBeads, Miltenyi
Biotec), magnetic bead depletion of non-T cells, Ficoll based
methods (e.g. RosetteSep.RTM., Stem Cell Technologies) or whole
blood isolation methods (MACSxpress.RTM., Miltenyi Biotec), as well
as panning on antibody coated surfaces.
EMBODIMENTS
[0088] In one embodiment of the present invention a first
nanomatrix of 1 to 500 nm, preferentially 10 to 200 nm in size
consists of a mobile matrix of a polymer of dextran and has
attached thereto one agent, e.g. anti CD3 mAb. A second nanomatrix
of 1 to 500 nm, preferentially 10 to 200 nm in size consists of a
mobile matrix of a polymer of dextran and has attached thereto
another agent, e.g. anti CD28mAb. In this case the nanomatrix of
the present invention is a nanomatrix wherein at least one first
agent and one second agent are attached to separate mobile
matrices.
[0089] A mixture of these nanomatrices is contacted with T cells,
thereby activating and inducing the T cells to proliferate.
[0090] Fine-tuning of nanomatrices for the stimulation of the T
cells is easily performed due to the high ratio of nanomatrices to
cells (normally larger than 500:1).
[0091] In another embodiment of the present invention a nanomatrix
of 1 to 500 nm, preferentially 10 to 200 nm in size consists of a
mobile matrix of a polymer of dextran and has attached thereto one
agent, e.g. anti CD3 mAb. In this case the nanomatrix of the
present invention is a nanomatrix wherein at least one first agent
is attached to mobile matrices. This nanomatrix is contacted with T
cells, thereby activating and inducing the T cells to
proliferate.
[0092] A second or more (multiple) co-stimulating agents, e.g. anti
CD28mAb, may be added as soluble agents to optimize or support the
activation induced by the nanomatrix with the first agent attached
thereto.
[0093] In another embodiment of the present invention a nanomatrix
of 1 to 500 nm, preferentially 10 to 200 nm in size consists of a
mobile matrix of a polymer of dextran and has attached thereto two
agents which provide activation signals to the cell, e.g. anti
CD3mAb and anti CD28 mAb. In this case the nanomatrix of the
present invention is a nanomatrix wherein at least one first agent
and one second agent are attached to the same flexible matrix.
[0094] Nanomatrices of this kind are contacted with T cells,
thereby activating and inducing the T cells to proliferate.
[0095] In another embodiment of the present invention a nanomatrix
of 1 to 500 nm, preferentially 10 to 200 nm in size consists of a
mobile matrix of a polymer of dextran and has attached thereto
multiple agents which provide activation signals to the cell, e.g.
anti-CD3mAb and anti-CD28 mAb, anti ICOS, anti-CD137 or other known
co-stimulatory molecules. In this case the nanomatrix of the
present invention is a nanomatrix wherein at least one first agent
and multiple other agents are attached to the same mobile
matrix.
[0096] Nanomatrices of this kind are contacted with T cells,
thereby activating and inducing the T cells to proliferate.
[0097] In another embodiment of the present invention a nanomatrix
of 1 to 500 nm, preferentially 10 to 200 nm in size consists of a
mobile matrix of a polymer of dextran and has attached thereto one
or more agents which provide activation signals to the cells, e.g.
anti CD3mAb and/or anti CD28 mAb. In addition the nanomatrix
carries magnetic, paramagnetic or superparamagnetic nano-crystals,
embedded into the polymer.
[0098] The nanomatrix is contacted with T cells, thereby activating
and inducing the T cells to proliferate. Optionally, after
stimulating of T cells the unbound magnetic, paramagnetic or
superparamagnetic nanomatrix may be removed by applying a magnetic
field gradient. Alternatively, the cells labelled with the magnetic
nanomatrices may be separated by applying a magnetic field
gradient, in particular a high-gradient magnetic field, and
subsequent expansion of the purified T cells.
[0099] Although there is no need to remove the nanomatrix after
activation and proliferation of the population of T cells due to
their property of being biologically inert with regard to
alteration of the cell function one might optionally remove the
nanomatrix with mild washing conditions, which are sufficient to
wash way the nanomatrices from the cells or cell culture. The
nanomatrices can easily be diluted by repeated washing steps to
effective concentrations below the T cell activation threshold.
This optionally removing step is much easier performed with the
nanomatrices than with beads or microspheres well known in the
state of the art due to their small size. If the nanomatrix carries
magnetic, paramagnetic or superparamagnetic nano-crystals, embedded
into the polymer than optionally the removal step can be performed
by applying a magnetic field gradient to the cell/nanomatrix
mixture.
[0100] The method of the invention can be used to expand selected T
cell populations for use in treating an infectious disease or
cancer. The resulting T cell population can be genetically
transduced and used for immunotherapy or can be used for in vitro
analysis of infectious agents such as HIV. Proliferation of a
population of CD4+ cells obtained from an individual or patient,
e.g. infected with HIV, can be achieved and the cells rendered
resistant, e.g. to HIV infection. Following expansion of the T cell
population to sufficient numbers, the expanded T cells are
re-infused into the individual or patient. Similarly, a population
of tumor-infiltrating lymphocytes can be obtained from an
individual afflicted with cancer and the T cells stimulated to
proliferate to sufficient numbers and restored to the individual.
In addition, supernatants from cultures of T cells expanded in
accordance with the method of the invention are a rich source of
cytokines and can be used to sustain T cells in vivo or ex
vivo.
[0101] In another embodiment of the present invention a nanomatrix
as described in any proceeding embodiment may be used in a closed
cell culture system, e.g. the sample processing system of
WO2009072003 or cell cultivation bags (Miltenyi Biotec, Baxter,
CellGenics), G-Rex devices (Wilson Wolf manufacturing), WAVE
Bioreactors (GE Healthcare), Quantum Cell Expansion System (Terumo
BCT). The nanomatrices have optimal connectivity to such a closed
cell culture system, they can be easily sterile filtrated and
integrated into the closed cell culture system. They ease the
processes of the closed cell culture system, i.e. stimulation of
the T cells or other target cells) because no removal of the
nanomatrices after the stimulation (and expanding) process is
necessary as described herein. The nanomatrix can be added in
relation to the volume of the culture rather than the T cell
number. Culture volume can be assesses by a balance or a camera
system (e.g. in the sample processing system of WO2009072003). The
use of the method of the present invention within a closed cell
culture system such as the sample processing system of WO2009072003
results in a safe and easy way to produce a pharmaceutical
composition of stimulated T cells due to the reduced risk of e.g.
contaminating agents such as other eukaryotic cells, bacteria or
viruses (safer and faster handling by the operator).
[0102] In another embodiment of the present invention cultivation
of T cells by use of the nanomatrix is performed within a magnetic
field. In this embodiment the nanomatrix consists of
superparamagnetic cores embedded into the mobile matrix. When
applied to a magnetic field magnetic forces are induced in the
nanomatrix and ligands triggering activating T cell receptors can
be concentrated for improved induction of proliferation.
Cultivation of T cells within a magnetic field can be performed in
a column consisting of a ferromagnetic matrix (e.g. of
WO2009072003) or in bag, flask or chamber in close proximity to
strong permanent magnet.
[0103] The present invention has broad applicability to any cell
type having a cell surface moiety that may be stimulated. In this
regard, many cell signalling events can be enhanced by the method
of the present invention. Such methodologies can be used
therapeutically in an ex vivo setting to activate and stimulate
cells for infusion into a patient or could be used in vivo, to
induce cell signalling events on a target cell population.
Preferentially the target cells of the method are T cells, but are
in no way limited thereto.
[0104] Prior to stimulation of T cells by the present invention the
T cells may be directly identified and/or separated or isolated
from blood, peripheral mononuclear blood cells (PBMC), body tissue
or cells from tissue fluids. The cells are normally identified
and/or separated from cell samples from mammals such as humans,
mouse, or rat, but especially from humans and preferably from test
subject and/or patients. The separation is performed by well known
sorting methods in the art. This includes for example affinity
chromatography or any other antibody-dependent separation technique
known in the art. Any ligand-dependent separation technique known
in the art may be used in conjunction with both positive and
negative separation techniques that rely on the physical properties
of the cells. An especially potent sorting technology is magnetic
cell sorting. Methods to separate cells magnetically are
commercially available e.g. from Invitrogen, Stemcell Technologies,
Cellpro, Advanced Magnetics, or Miltenyi Biotec. In addition to
mixtures of T cells with other cells, such as monocytes,
macrophages, dendritic cells, B cells or other cells which are part
of hematologic cell samples, such as blood or leukapheresis, highly
purified T cell populations can be used for contacting with the
presented invention, including T cell subpopulations, such as CD4+
T cells, CD8+ T cells, NKT cells, .gamma./.delta. T cells,
.alpha./.beta. T cells, CD4+CD25+Foxp3+ regulatory T cells, naive T
cells (CD45RA+CCR7+ and/or CD62L+) or central memory T cells
(CD45R0+CCR7+), effector memory T cells (CD45R0+CCR7-) or terminal
effector T cells (CD45RA+CCR7-). Nanomatrices provide sufficient
crosslinking activity to the T cell receptor, therefore additional
crosslinking, e.g. via Fc-receptor expressing cells such as
monocytes or dendritic cells is not required for activation.
[0105] Target cell populations, such as the T cell populations
obtained via the present disclosure may be administered either
alone, or as a pharmaceutical composition in combination with
diluents and/or with other components such as IL-2 or other
cytokines or cell populations. Briefly, pharmaceutical compositions
of the present disclosure may comprise a target cell population as
described herein, in combination with one or more pharmaceutically
or physiologically acceptable carriers, diluents or excipients. A
pharmaceutical composition may comprise a) a population of T cells,
wherein said T cells are proliferated to therapeutically effective
amounts according to the present invention; and b) one or more
pharmaceutically or physiologically acceptable carriers, diluents
or excipients. Such a composition may contain traces of
nanomatrices which are biologically inert with regard to alteration
of the cell function but may be biodegradable and which are
non-toxic and non-antigenic to humans.
[0106] Compositions of the present disclosure are preferably
formulated for intravenous administration.
[0107] Pharmaceutical compositions of the present disclosure may be
administered in a manner appropriate to the disease to be treated
(or prevented). The quantity and frequency of administration will
be determined by such factors as the condition of the patient, and
the type and severity of the patient's disease, although
appropriate dosages may be determined by clinical trials.
EXAMPLES
Example 1
Preparation of Nanomatrices
[0108] Magnetic nanomatrices were produced by a modification of the
procedure of Molday and MacKenzie. Ten grams of Dextran T40
(Pharmacia Uppsala, Sweden), 1.5 g FeCl.sub.3.6H.sub.2O and 0.64 g
FeCl.sub.2.4H.sub.2O are dissolved in 20 ml H.sub.2O, and heated to
40.degree. C. While stirring, 10 ml 4N NaOH are added slowly and
the solution is heated to 70.degree. C. for 5 min. The particle
suspension is neutralized with acetic acid. To remove aggregates
the suspension is centrifuged for 10 min at 2,000 g and filtrated
through a 0.22 .mu.m pore-size filter (Millex GV, Millipore,
Molsheim, France). Unbound Dextran is removed by washing in a
high-gradient magnetic field (HGMF). HGMF washing of magnetic
nanomatrices is performed in steelwool columns made as described
below and placed in a magnetic field of approx. 0.6 Tesla (MACS
permanent magnet, Miltenyi Biotec GmbH, Bergisch Gladbach,
Germany). Ten milliliters of nanomatrix suspension are applied to a
15.times.40 mm column of 2 g steelwool. The loaded column is washed
with 30 ml 0.05 M sodium acetate. After removing the column from
the external magnetic field, the magnetic nanomatrices are eluted
with 0.05 M sodium acetate. The nanomatrices form a brown
suspension. The relative particle concentration is given as optical
density at 450 nm. The size of the nanomatrices was determined by
electron microscopy and dynamic light scattering to be 30.+-.20 nm
(e.m.) and 65.+-.20 nm (DLS). Dextran content and magnetite content
of the matrix within the colloidal solution were determined to be
in the range of about 3.5 mg/ml and 4.8 mg/ml, respectively,
resulting approximately in a 40:60 w/w ratio. Based on the density
of the dried nanomatrix of 2.5 g/mL determined by pycnometer and
the known densities of dextran and magnetite of 1.6 g/ml and 5.2
g/ml, respectively, the volume of dextran can be calculated to be
about 70% for the dried nanomatrix. The nanomatrices show
superparamagnetic behavior, as determined by susceptibility
measurements. The size of the trapped ferrit microcrystals was
determined from magnetic measurements to be approximately 10
nm.
[0109] CD3 antibodies (clone OKT3) and CD28 antibodies (clone15E8)
(Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) were conjugated
to the same or separate nanomatrices by standard bioconjugation
chemistry (Bioconjugate Techniques, 2nd Edition, By Greg T.
Hermanson, Published by Academic Press, Inc., 2008).
Example 2
Expansion of T Cells Using Nanomatrices at Various CD3/CD28
Concentrations and Ratios Versus CD3/CD28 MACSiBeads
[0110] The current state-of-the-art reagents for activation of
highly purified T cells comprise activating antibodies against
CD3/CD28 immobilized either on the surfaces of a cell culture dish
or large cell-sized (4-5 .mu.m) particles. Both techniques are
error prone and technically difficult to realize and standardize,
especially under GMP-compatible production conditions. In contrast
nanomatrices can be easily prepared and conveniently be used for
cell culture under GMP-conditions. Therefore we compared the T cell
activation potential by analysing the expansion potential of the
CD3/CD28 coated nanomatrices at various concentrations and CD3/CD28
ratios with commercially available cell stimulation beads
(MACSiBeads, o 4.5 .mu.m, Miltenyi Biotec GmbH). As can be seen in
FIG. 1 the nanomatrices expand T cells efficiently even at very low
CD3 concentrations (20-100 ng/ml) which are also typically used for
soluble CD3/CD28 in the presence of accessory cells which provide
crosslinking. Besides the antibody concentration the CD3/CD28 ratio
can also influence the cell activation and provides an additional
means to optimize the T cell culture. The expansion at optimal
doses (20-300 ng/ml) was similar or better than the standard
reagent (MACSiBeads). At higher doses the expansion was reduced due
to overstimulation of the T cells (activation-induced cell death),
a phenomenon known to occur at a too high degree of TCR
stimulation. Taken together, these results show that CD3/CD28
coated nanomatrices can efficiently activate and expand T cells at
very low antibody concentrations and without the need for
additional crosslinking.
Example 3
Comparison CD3/CD28 Conjugated to Nanomatrices Versus 200 nm and
300 nm Solid Particles
[0111] As outlined above currently available reagents for
activation of T cells can be split into two groups. Soluble
antibodies stimulating, e.g. against CD3 and CD28, require
immobilisation either on a surface of the cell culture dish or via
receptors an accessory cells, e.g. Immunoglobulin Fc-receptors.
Reagents which do not depend on an extra crosslinking step to be
used for T cell activation, e.g. to stimulate highly purified T
cells in the absence of accessory cells, are based on cell-sized
particles (o 4-5 .mu.m) coated with stimulating CD3 and CD28
antibodies. It is known that solid particles below a critical
diameter of about 1 .mu.m are not suitable to properly expand T
cells. To show the unique activating capacity of the CD3/CD28
coated nanomatrices (o 50-200 nm), we compared their activating
capacity with solid particles of similar size (200 nm, and 300 nm)
versus cell sized particles (o 4.5 .mu.m). Since small solid
particles do not usually lead to expansion of T cells we analysed
early T cell activation markers (CD25 up-regulation and loss of CD3
expression) to have a sensitive screen for T cell activation. CD25
is up-regulated within the first 24-48 hours following T cell
stimulation. Because the TCR induced CD25 up-regulation is further
supported by IL-2, we also added IL-2 to the culture conditions to
maximize the sensitivity of the assay. Another direct result of TCR
stimulation is the downregulation of the T cell receptor, which can
be analysed via loss of CD3 expression on the cell surface. Highly
purified T cells were cultured with CD3/CD28 coated nanomatrices
(100 ng/ml CD3) or solid particles with a diameter of 4.5 .mu.m
(MACSiBeads) or 200 nm (Ademtech beads) both covalently coated with
CD3 and CD28 antibodies. MACSiBeads were used at an optimal 1:1
ratio whereas 200 nm particles were titrated to achieve an active
dose of CD3 and CD28 ranging from 25-3000 ng/ml CD3. On day 3 and 5
the frequency of CD25+ T cells and on day 3 the expression
intensity of CD3 was measured.
[0112] As can be seen in FIGS. 2A, B and C nanomatrices lead to
strong activation at the optimal dose (100 ng/ml) as shown by
up-regulation of CD25 [FIG. 2B (day 3), FIG. 2C (day 5)] and
downregulation of CD3 (FIG. 2A) which occurred at similar levels
like with the cell-sized MACSiBeads. In sharp contrast no CD25
up-regulation and almost no CD3 down-regulation was seen for 200 nm
solid particles even at 30 fold higher CD3/CD28 concentrations.
Even on day five 200 nm solid particles were not able to induce
CD25 expression to a similar level like the nanomatrix. Only at
high concentrations (5-30 fold higher than for the nanomatrix)
there was a slight upregulation observed achieving about 50-70% of
the levels of the nanomatrix.
[0113] These data show that despite their small size mobile
nanomatrices indeed have a unique potential to activate T cells
when compared to similarly sized particles with a solid surface.
The titration experiment also shows that the lack of activation by
CD3/CD28 coated 200-300 nm-sized solid particles cannot simply be
compensated by higher doses of particles but obviously there is a
different quality of activation signal induced by the
nanomatrix.
Example 4
Expansion of Purified T Cell Subsets
[0114] As indicated above various T cell subsets can have different
activation requirements. In particular naive T cells are difficult
to activate in the absence of accessory cells. Furthermore CD4 and
CD8 T cells may have different needs when activated alone or in
presence of additional cell types. To show that all T cell subsets
can be expanded equally well by nanomatrices, we activated purified
CD4 and CD8 naive T cells, total naive T cells or total T cells
with either nanomatrices at the indicated dose and composition or
MACSiBeads and compared their expansion. As shown in FIG. 3 all
subsets can be efficiently expanded by nanomatrices and at
comparable level to the standard MACSiBead culture.
Example 5
Expansion of CD25+Foxp3+ Regulatory T Cells (Treg)
[0115] Treg are of particular interest for therapeutic applications
for transplantation, autoimmunity and chronic inflammation and Treg
are difficult to expand in vitro without loss of regulatory
activity, i.e. Foxp3 expression. Therefore we also analysed whether
CD25 selected Treg cells (Foxp3 purity typically 60-90%) from
various donors can be expanded using nanomatrices. To support
growth of Treg versus conventional T cells the expansion was done
in the presence of 100 nM Rapamycine, a well described drug
inhibiting conventional T cell growth. As shown in FIG. 4 following
14 days of culture Treg can be expanded 10-20 times (w/o Rapa) or
5-10 times (with Rapa). As described before without Rapa the Foxp3
purity was highly variable (10-75%) whereas in the presence of Rapa
the purity was always >50%. Taken together these results show
that nanomatrices can even be used to activate and expand Treg in
culture.
Example 6
Comparison of T Cell Activation by CD3/CD28-Conjugated to the Same
Nanomatrix Versus CD3 and CD28 Conjugated to Separate
Nanomatrices
[0116] It is described in various applications of CD3 and CD28
based T cell activation reagents that both antibodies have to be
immobilized onto the same surface for optimal activation. Therefore
we also tested whether this is also required for CD3 and CD28
conjugated to nanomatrices. We compared expansion of purified naive
T cells activated by a CD3/CD28 nanomatrix versus CD3
nanomatrix+CD28 nanomatrix mixed at different
ratios/concentrations. Expansion (day 5) and cell division (day 7),
as measured by Violetye dilution was analysed. As shown in FIG. 5A,
B the stimulation with the CD3 nanomatrix alone did not induce
significant expansion and only few cell divisions can be observed,
as it is expected for naive T cells which depend on a costimulatory
signal. However addition of the CD28 nanomatrix, already at 10-50
ng/ml induced full cell dividing activity and also expansion of T
cell numbers, which was similar to the CD3/CD28 control nanomatrix
or the standard MACSiBeads. These data clearly show that both
antibodies may be conjugated to separate nanomatrices, which can be
mixed hereafter for optimised use. This facilitates the production
process and quality control of the single nanomatrices and improves
the flexibility of the reagent, e.g. facilitating the optimisation
of the activation conditions for specialised T cell subsets by
titrating various CD3 and CD28 concentrations and ratios
(fine-tuning).
Example 7
The Effect of Conjugation of Soluble CD3 or CD28 to the
Nanomatrix
[0117] To rule out the possibility that similar results as with the
CD3 and/or CD28 coated nanomatrix could be achieved by use of the
respective soluble antibodies, we compared the stimulating effects
of CD3 or CD28 coated the nanomatrix with soluble antibodies at
various concentrations to demonstrate that indeed the conjugation
of the antibodies to the matrix is the critical step to obtain good
T cell activation. IL-2 was added to all cultures. As shown in FIG.
6A a soluble CD3 alone did not induce any significant up-regulation
of the early activation markers CD25 and CD69 in naive T cells over
a wide concentration range (10-10000 ng/ml) whereas CD3 coated
Nanomatrix (100 ng/ml CD3) induced CD25/CD69 expression in 20-60%
of the cells. In the presence of a saturating amount (200 ng/ml) of
soluble CD28 as costimulator (FIG. 6B) soluble CD3 also induced
CD25/CD69 expression in about 20-40% of the cells at the highest
tested doses (100-10000 ng/ml). In contrast the CD3 coated
nanomatrix (100 ng/ml CD3) induced CD25/CD69 expression in 40-70%
of the cells.
[0118] We also tested the effect of conjugation of CD28 antibodies
to the nanomatrix. Since the effects of costimulation are best
visualized under suboptimal CD3 stimulation, we titrated CD28
either soluble or conjugated to the nanomatrix in the presence of
soluble CD3 to a culture of naive T cells. As shown in FIG. 6C
soluble CD3 alone similar to the induction of CD25/CD69 as shown
above did not induce any expansion of the naive T cells. In the
presence of soluble CD28 however a 2-6 fold expansion was
detectable but only at the highest tested dose of CD28 (10000
ng/ml). In contrast to this, CD28 conjugated to nanomatrix induced
a similar degree of expansion already at a 1000 fold lower
concentration (10 ng/ml). These data show again the strong
crosslinking and T cell activating capacity of nanomatrix versus
soluble antibodies which explains why CD3CD28 conjugated
nanomatrices in contrast to soluble antibodies can be used to
activate and expand even naive human T cells in vitro.
Example 8
Nanomatrices can be Used to Activate T Cells for Introduction of
TCR Genes by Viral Transduction
[0119] One important application for activating and expanding T
cells and in particular purified cell subsets is their genetic
manipulation, e.g. to introduce a certain antigen receptor with
specificity for tumor antigens. We have used nanomatrices to
activate purified naive (T.sub.N, CD62L+CD45RA+), central memory
(T.sub.CM, CD62L+CD45RA-) and effector (T.sub.EM, CD62L-CD45RA-) T
cells and transduced them using a retroviral vector expressing a
TCR specific for MART-1, a tumor antigen. To test the relative
frequency of transgene expression in these T cell subsets we
performed MHC-peptide Class I tetramer staining. All T cell subsets
are efficiently transduced (>50%) independent on the stimulatory
conditions we tested (FIG. 7). We also compared the in vitro
expansion of the transduced T cells. As shown in FIG. 8 after 10
days we observed no differences with regard to expansion of the
three subsets under all conditions. All activation regimens for the
isolated T cell subsets were equal or better to the "standard"
stimulation of total PBMC with soluble CD3 (all values were
normalized to this standard to allow better comparison between
different donors). We observed a trend (not statistically
significant) for better expansion when T cell subsets are
stimulated with MACSiBeads or nanomatrices when compared to coated
.alpha.CD3+.alpha.CD28. We further investigated the functional
activity of the introduced MART-1 TCR and the differentiation
status of transduced cells looking at surface markers and cytokine
production upon restimulation with a MART-1+HLA-A2+ tumor cell
line. As shown in FIG. 9 nanomatrix- and MACSiBead-stimulated
T.sub.CM and T.sub.N cells seem to have a higher expression of
CD62L and CCR7, two molecules facilitating migration of the T cells
into peripheral lymph nodes. This capacity is regarded as
beneficial to promote long term persistence and functional activity
of transferred T cells in vivo and thus is thought to increase
therapeutic efficacy. The percentage of MART-1 reactive
IFN.gamma..sup.+ cells tend to be higher in T.sub.CM and T.sub.EM
CD8.sup.+ T cell subsets compare to T.sub.N in all stimulatory
conditions but this was not statistically significant (FIG. 10 top
panel). Focussing on the IL-2 production (FIG. 10 middle panel) we
observed that a higher percentage of T.sub.N cells produces IL-2
when they have been stimulated with MACSiBeads/nanomatrices when
compared to coated .alpha.CD3+.alpha.CD28 stimulation. The same is
true for TNF.alpha. producing cells detected in T.sub.N subset when
stimulated with MACSiBeads (FIG. 10 bottom panel). These results
indicate cells of T.sub.N derived cells stimulated with beads
showed diminished effector cell differentiation, suggesting less
progress toward terminal differentiation.
[0120] Taken together, the results indicate that CD3/CD28
nanomatrices can be used to efficiently activate and transduce
purified T cell subsets to generate fully functional T cell
transplants, e.g. for tumor therapy.
* * * * *