U.S. patent application number 14/377825 was filed with the patent office on 2015-01-08 for pepmixes to generate multiviral ctls with broad specificity.
The applicant listed for this patent is Baylor College of Medicine. Invention is credited to Ulrike Gerdemann, Ann Marie Leen, Cliona M. Rooney, Juan Fernando Vera Valdes.
Application Number | 20150010519 14/377825 |
Document ID | / |
Family ID | 48948057 |
Filed Date | 2015-01-08 |
United States Patent
Application |
20150010519 |
Kind Code |
A1 |
Leen; Ann Marie ; et
al. |
January 8, 2015 |
PEPMIXES TO GENERATE MULTIVIRAL CTLS WITH BROAD SPECIFICITY
Abstract
The present invention concerns methods of generating CTLs that
are able to target at least one antigen from two or more viruses.
The method includes exposing mixtures of peptides for different
antigens to the same plurality of PBMCs and, at least in certain
aspects, expanding the cells in the presence of IL4 and IL7.
Inventors: |
Leen; Ann Marie; (Bellaire,
TX) ; Valdes; Juan Fernando Vera; (Bellaire, TX)
; Rooney; Cliona M.; (Bellaire, TX) ; Gerdemann;
Ulrike; (Cambridge, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Baylor College of Medicine |
Houston |
TX |
US |
|
|
Family ID: |
48948057 |
Appl. No.: |
14/377825 |
Filed: |
February 8, 2013 |
PCT Filed: |
February 8, 2013 |
PCT NO: |
PCT/US13/25342 |
371 Date: |
August 8, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61596875 |
Feb 9, 2012 |
|
|
|
Current U.S.
Class: |
424/93.71 ;
506/26 |
Current CPC
Class: |
C12N 2501/2307 20130101;
C12N 2501/2315 20130101; A61K 2039/57 20130101; A61K 2039/5158
20130101; A61K 2039/55527 20130101; C12N 7/00 20130101; A61K
2039/577 20130101; A61K 39/12 20130101; C12N 2501/2304 20130101;
Y02A 50/467 20180101; Y02A 50/30 20180101; C12N 2501/2302 20130101;
A61K 2039/572 20130101; C12N 5/0638 20130101; A61K 2039/70
20130101; C12N 2710/16134 20130101; C12N 2710/16234 20130101; Y02A
50/394 20180101 |
Class at
Publication: |
424/93.71 ;
506/26 |
International
Class: |
C12N 5/0783 20060101
C12N005/0783; C12N 7/00 20060101 C12N007/00; A61K 39/12 20060101
A61K039/12 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with government support under grants
U54 HL081007 and N01-HB-10-03 awarded by National Institutes of
Health. The government has certain rights in the invention.
Claims
1. A method of generating cytotoxic T-lymphocytes (CTLs) that
target at least one antigen from two or more viruses, comprising
the steps of: contacting a plurality of peripheral blood
mononuclear cells with at least two libraries of peptides, said
libraries of peptides each comprising peptides that correspond to a
particular viral antigen; and expanding the plurality of cells in
the presence of one or more cytokines.
2. The method of claim 1, wherein said method occurs in the absence
of exposing the libraries to isolated peptide-pulsed dendritic
cells prior to expanding the CTLs.
3. The method of claim 1, wherein the one or more cytokines are
selected from the group consisting of IL4, IL7 and a combination
thereof.
4. The method of claim 1, wherein the peptides are further defined
as peptides that overlap in sequence to span part or all of a viral
antigen.
5. The method of claim 3, wherein the peptides overlap by at least
three amino acids.
6. The method of claim 3, wherein the peptides are at least seven
amino acids in length.
7. The method of claim 1, wherein the viruses are selected from the
group consisting of EBV, CMV, Adenovirus, BK virus, HHV6, RSV,
Influenza, Parainfluenza, Bocavirus, Coronavirus, LCMV, Mumps,
Measles, Metapneumovirus, Parvovirus B, Rotavirus, West Nile Virus,
JC, HHV7, and a combination thereof.
8. The method of claim 1, wherein the virus is EBV and the antigen
is selected from the group consisting of EBNA1, LMP2, and
BZLF1.
9. The method of claim 1, wherein the virus is CMV and the antigen
is selected from the group consisting of IE1 and pp65.
10. The method of claim 1, wherein the virus is Adv and the antigen
is selected from the group consisting of Hexon and penton.
11. The method of claim 1, wherein the virus is BK virus and the
antigen is selected from the group consisting of LT and VP-1.
12. The method of claim 1, wherein the virus is HHV6 and the
antigen is selected from the group consisting of U14, U11, U71,
U54, and U90.
13. The method of claim 1, wherein the virus is RSV and the antigen
is selected from the group consisting of N and F.
14. The method of claim 1, wherein the virus is Influenza and the
antigen is selected from the group consisting of MP1 and NP1.
15. The method of claim 1, wherein the CTLs are administered to an
individual.
16. The method of claim 1, wherein the CTLs are administered to an
immunocompromised individual.
17. The method of claim 15, wherein the individual has had
allogeneic stem cell transplant.
18. The method of claim 14, wherein the cells are administered by
injection.
19. The method of claim 17, wherein the injection is
intravenous.
20. The method of claim 1, wherein the CTLs are further defined as
polyclonal CD4+ and CD8+ CTLs.
21. The method of claim 14, wherein the PBMCs are allogeneic to the
individual.
22. The method of claim 14, wherein the PBMCs are autologous to the
individual.
23. The method of claim 1, further comprising the step of exposing
the CTLs to one or more compositions that stimulate cell division.
Description
[0001] This application claims priority to U.S. Provisional Patent
Application Ser. No. 61/596,875, filed Feb. 9, 2012, which is
incorporated by reference herein in its entirety.
TECHNICAL FIELD
[0003] The present invention generally concerns the fields of
immunology, cell biology, molecular biology, and medicine.
BACKGROUND OF THE INVENTION
[0004] Although hematopoietic stem cell transplant (HSCT) may cure
hematological malignancies and genetic disorders, extension to
donors other than HLA-matched siblings has resulted in the
emergence of viral infections as major contributors to
post-transplant morbidity and mortality.sup.1-4. With the advent of
more intensive viral screening and improved detection, increasing
numbers of viral pathogens have been implicated in these
complications, expanding from cytomegalovirus (CMV), Epstein-Barr
virus (EBV), herpes-simplex virus (HSV), Adenovirus (Adv), and BK
to include human herpesvirus (HHV)-6, Respiratory Syncytial virus
(RSV), parainfluenza, and influenza.sup.2. While pharmacological
agents are standard therapy for some, they have substantial
toxicities, generate resistant variants, are frequently ineffective
and do not provide long-term protection.sup.5,6.
[0005] Restoration of virus-specific immunity offers an attractive
alternative to conventional drugs. The inventors have shown that in
vitro expanded virus-specific cytotoxic T lymphocytes (CTL)
generated from stem cell donors with specificity for one (EBV), two
(EBV and Adv) or three (EBV, CMV and Adv) viruses are safe and
effectively prevent and treat viral infection or disease in the
HSCT setting.sup.7-9. More recently, banked, partially HLA-matched
virus-specific CTL (3rd party CTLs) are showing promise in
allograft recipients with advanced viral disease.sup.10-12.
[0006] Despite these encouraging clinical results broader
implementation of T cell therapy is restricted by (i) the limited
spectrum of viruses that can be effectively targeted in a single T
cell line, and (ii) the logistics of manufacture. Antigenic
competition between high and low frequency T cells as well as
between multiple antigens expressed at different levels and
competing for presentation on shared antigen presenting cells
(APCs) may favor generation of lines dominated by responses to a
single virus or to a restricted spectrum of viral
antigens.sup.13,14, thus limiting the antiviral coverage provided
by a single T cell product. In addition, our current manufacturing
process is complex, requiring infectious virus material (EBV/Adv),
production of a clinical grade vector, and prolonged (10-12 weeks)
in vitro culture.sup.8-10,15. To address this latter problem some
groups have evaluated more rapid approaches for producing T cell
products for adoptive transfer. These include streptamer selection
to directly isolate virus-specific CD8+ T cells from peripheral
blood.sup.16, as well as the selection of cells based on cytokine
production (IFN) or expression of activation markers (e.g. CD154)
following antigen exposure.sup.17-19. However, these approaches are
expensive, require a large starting blood volume, which is not
always available, particularly in the matched unrelated donor
setting, and cannot be applied to viruses with low circulating T
cell precursor frequencies.
[0007] There is a need in the art for a mechanism by which one can
rapidly generate a single preparation of polyclonal CTLs that is
consistently specific for immunodominant and/or subdominant
antigens derived from more than one virus, including those that are
frequent causes of post-transplant disease or death, for
example.
BRIEF SUMMARY OF THE INVENTION
[0008] The present invention is directed to methods and
compositions that concern immune system components that are
modified to immunogenically recognize particular targets. In some
embodiments, the present invention concerns the development of
cytotoxic T-lymphocytes (CTLs) that target a biological moiety that
elicits an immune response in an individual. In specific
embodiments, the present invention concerns the development of CTLs
that target at least one antigen from a pathogen (including viral,
bacterial, or fungal) or other disease-associated antigen. In
certain aspects of the invention, the present invention concerns
the development of CTLs that target antigens from at least one
virus, for example. In alternative embodiments, the present
invention concerns the development of CTLs that target at least one
tumor antigen, for example. In at least some cases, the CTLs target
antigens from two or more viruses (or two or more tumors, in
alternative embodiments). In some embodiments, the CTLs target one
or more, two or more, three or more, or four or more antigens from
the same virus. In some embodiments, the CTLs target one antigen
from more than one virus. In certain embodiments, the CTLs target
one or more, two or more, three or more, or four or more antigens
from different viruses.
[0009] The present invention provides significant and non-obvious
improvements on methods for generating CTL lines with specificity
against multiple tumor antigens or multiple viruses (for example).
In the generation of CTLs with such specificity, the present
invention obviates the need for dendritic cells in the preparation
of such lines. In some cases, the antigen is presented to PBMCs in
the form of one or more peptides that span some or all of the
antigen. The antigenic peptides may be provided to the PBMCs in a
library of peptide mixtures, which may be referred to as pepmixes.
In other aspects of the invention, in the preparation of the CTLs
the invention allows for the pooling of a variety of pepmixes. In
some cases, the collection of antigens may include both
immunodominant and subdominant antigens, yet despite the presence
of immunodominant antigens in the collection with subdominant
antigens, CTLs specific antigens including subdominant antigens are
surprisingly generated.
[0010] In some embodiments of the invention, an individual is in
need of the methods and/or compositions of the invention. In
specific embodiments, the individual is immunocompromised (which
for example, may be defined as an individual whose ability to fight
infectious disease or cancer with the immune system is compromised
or entirely absent). In specific embodiments, the immunocompromised
individual has had a stem cell transplant, has had an organ
transplant and/or has received one or more cancer treatments,
including chemotherapy or radiation, for example. In some cases,
the individual acquired or inherited immune deficiency disorder. In
some embodiments, those that are immunocompromised by their disease
and/or its treatment are provided methods and/or compositions if
the invention.
[0011] In some embodiments of the invention, there is a mechanism
by which one can rapidly generate a single preparation of
polyclonal (for example, CD4+ and CD8+) CTLs that are consistently
specific for a variety of immunodominant and/or subdominant
antigens derived from one or more viruses (for example, EBV, CMV,
Adv, BK virus, HHV6, RSV and Influenza) that are frequent causes of
post transplant disease or death. The invention is readily
adaptable to clinical implementation and is useful as an "off the
shelf" broad spectrum antiviral agent. The invention uses
standardized (synthetic) peptides as a stimulus and enhancement of
cytokines to promote the survival and expansion of T cells, is
readily adaptable to clinical implementation, and is useful as a
safe and effective broad spectrum antiviral agent for all high risk
transplant recipients, for example.
[0012] In some embodiments of the invention, there is a method of
generating cytotoxic T-lymphocytes (CTLs) that target at least one
antigen from two or more viruses, comprising the steps of:
contacting a plurality of peripheral blood mononuclear cells with
at least two libraries of peptides, said libraries of peptides each
comprising peptides that correspond to a particular viral antigen;
and expanding the plurality of cells in the presence of one or more
cytokines. In specific embodiments, the method occurs in the
absence of exposing the libraries to isolated peptide-pulsed
dendritic cells prior to expanding the CTLs. In certain
embodiments, the one or more cytokines are selected from the group
consisting of IL4, IL7 and a combination thereof. In some
embodiments, the peptides are further defined as peptides that
overlap in sequence to span part or all of a viral antigen. For
example, in certain aspects the peptides overlap by at least three,
four, five, or six amino acids, and in some embodiments the
peptides are at least six, seven, or eight or more amino acids in
length.
[0013] In some embodiments of the invention, there viruses targeted
in the invention are selected from the group consisting of EBV,
CMV, Adenovirus, BK virus, HHV6, RSV, Influenza, Parainfluenza,
Bocavirus, Coronavirus, LCMV, Mumps, Measles, Metapneumovirus,
Parvovirus B, Rotavirus, West Nile Virus, JC, HHV7, and a
combination thereof. In specific aspects, the virus is EBV and the
antigen is selected from the group consisting of EBNA1, LMP2, and
BZLF1. In specific aspects, the virus is CMV and the antigen is
selected from the group consisting of IE1 and pp65. In specific
cases, the virus is Adv and the antigen is selected from the group
consisting of Hexon and penton. In some embodiments, the virus is
BK virus and the antigen is selected from the group consisting of
LT and VP-1. In some embodiments, the virus is HHV6 and the antigen
is selected from the group consisting of U14 and U90. In specific
aspects, the virus is RSV and the antigen is selected from the
group consisting of N and F. In certain embodiments, the virus is
Influenza and the antigen is selected from the group consisting of
MP 1 and NP 1.
[0014] In at least some methods of the invention, the CTLs
generated thereby are administered to an individual, for example,
an immunocompromised individual. In some cases, the individual has
had allogeneic stem cell transplant. In specific embodiments, the
cells are administered by injection, such as intravenous,
intramuscular, intradermal, subcutaneous, intraperitoneal
injection, and so forth, for example. In some embodiments, the
individual has lymphoma or leukemia. In some embodiments, the CTLs
are further defined as polyclonal CD4+ and CD8+ CTLs. The PBMCs may
be allogeneic to the individual or are autologous to the
individual. In some embodiments, the methods of the invention
further comprise the step of exposing the CTLs to one or more
compositions that stimulate cell division, such as
phytohemagglutinin; in some aspects the compound is a mitogen.
[0015] The foregoing has outlined rather broadly the features and
technical advantages of the present invention in order that the
detailed description of the invention that follows may be better
understood. Additional features and advantages of the invention
will be described hereinafter which form the subject of the claims
of the invention. It should be appreciated by those skilled in the
art that the conception and specific embodiment disclosed may be
readily utilized as a basis for modifying or designing other
structures for carrying out the same purposes of the present
invention. It should also be realized by those skilled in the art
that such equivalent constructions do not depart from the spirit
and scope of the invention as set forth in the appended claims. The
novel features which are believed to be characteristic of the
invention, both as to its organization and method of operation,
together with further objects and advantages will be better
understood from the following description when considered in
connection with the accompanying figures. It is to be expressly
understood, however, that each of the figures is provided for the
purpose of illustration and description only and is not intended as
a definition of the limits of the present invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] For a more complete understanding of the present invention,
reference is now made to the following descriptions taken in
conjunction with the accompanying drawing, in which:
[0017] FIG. 1: Growth promoting cytokines enhance the activation
and expansion of antigen-specific CTLs. PBMC were stimulated with
pp65 pepmix in the presence of IL2, IL15, IL4+7 or without
exogenous cytokines. Cell expansion were evaluated after 9-11 days
of culture by cell counting using trypan blue exclusion (n=5).
Results are shown as mean cell numbers+/-SEM. (A). Panel B CD3+ T
cell proliferation in the different culture conditions as evaluated
by CFSE dilution. M1 shows the percentage of cells that underwent
at least 7 cell doublings on day 10 after stimulation. Bulk
cultures were analyzed for T and NK-cell marker expression on day
10 after activation. Mean expression+/-SEM in CTL lines generated
from 5 donors are shown in Panel C. Panel D shows cytokine
production from CD3/CD4+ (helper) and CD3/CD8+ (cytotoxic) CTLs on
day 9 after initiation in one representative donor (dot plots shown
were gated on CD3+ cells). Summary intracellular cytokine
production results from three donors (mean+/-STDEV) are shown in
Panel E. Finally the cytokine production profile of pp65-specific
CTL initiated with or without cytokines was evaluated by multiplex
assay using supernatant harvested 18 h after antigenic
restimulation (n=4). Th1 cytokines are shown in the left panel
while prototypic Th2 cytokines are shown in the right panel (Panel
F). Presence of regulatory T cells were evaluated by FoxP3
staining. Plots shown are gated on CD3+/CD4+ CTLs (Panel G).
[0018] FIG. 2: Peptide-stimulated and plasmid-activated CTLs share
similar phenotypic and functional characteristics. Panel A CTLs
were stimulated either directly with a pp65 pepmix or using DCs
nucleofected with a DNA plasmid encoding the same antigen. Cell
expansion was evaluated by counting using trypan blue exclusion
(n=4). Panel B shows the expression of cell surface markers
(average+/-STDEV expression) on CTLs 11 days after stimulation
(n=4). The breadth of T cell reactivity in plasmid and
pepmix-activated pp65-specific CTLs was evaluated by IFN ELIspot on
day 9 using a total of 22 mini peptide pools representing all pp65
peptides. Data were normalized to 100% for maximum number of SFC
per 1.times.10.sup.5 CTL. (Panel C). Panel D shows the TCR avidity
of plasmid vs. pepmix activated CTL generated from 2 representative
donors. To assess avidity pp65-CTLs were stimulated with serial
dilutions of pp65 pepmix (pp65) or relevant (HLA-matched) epitope
peptides (NLV, QAD). IFN release of stimulated CTLs was evaluated
by ELIspot assay and maximum SFC/1.times.10.sup.5 cells was
normalized to 100% for comparison purposes.
[0019] FIG. 3: Peptide length does not affect breadth of
reactivity. Panel A shows a schematic of three peptide libraries
spanning a portion of Adv-Hexon that were used for CTL initiation.
Peptide libraries consisted of 15aa, 20aa or 30aa peptides covering
the immunogenic C-terminal 414aa of Adv-Hexon. B Phenotypic
analysis of CTLs performed on day 10 after stimulation (n=6).
Results are shown as mean+/-SEM. Breadth of reactivity was tested
using IFN.gamma. ELIspot as a readout, with the 15mer Hexon
overlapping peptide library divided into mini-pools such that each
pool contained 5-6 contiguous peptides, as a stimulus.
[0020] FIG. 4: Pepmix-activated trivirus-specific CTL lines show
similar specificity to plasmid-activated T cells. CTL lines were
generated using DCs nucleofected with DNA plasmids encoding EBNA-1,
LMP2, BZLF-1 (EBV), Hexon, Penton (Adenovirus), IE-1 and pp65 (CMV)
or direct PBMC stimulation with the corresponding pepmixes.
Specificity was determined 10 days after initiation with IFN
ELIspot as readout. Results are expressed as SFC/1.times.10.sup.5
input cells. Control was IFN release in response to stimulation
with irrelevant pepmix.
[0021] FIG. 5: Generation of multivirus-specific CTLs. Panel A
shows a schematic of antigen pooling strategy for CTL initiation.
PBMCs were stimulated with pepmixes pooled by virus (A), divided
into sub-pools--immunodominant and subdominant (B), divided into
sub-pools encompassing antigens from latent or lytic viruses (C),
and finally all antigens were pooled together in a mastermix (D).
After activation PBMCs were pooled and transferred to the G-Rex10
(15.times.10.sup.6/G-Rex). After 10 days the specificity of the CTL
lines generated using these 4 pooling strategies were analyzed
using IFN ELIspot assay as readout and individual pepmixes as a
stimulus. Results from 2 representative donors are presented in
Panel B showing no difference in the specificity of lines. Panel C
confirms that multivirus CTL can be reproducibly generated by
pooling all pepmixes into one mastermix for activation (n=8).
Results are expressed as SFC/1.times.10.sup.5 input cells+/-SEM.
Control was IFN release in response to stimulation with an
irrelevant pepmix. Antigen specificity of CD3/CD8+ (cytotoxic) and
CD3+CD8- (helper) T cells was evaluated by intracellular IFN
staining after overnight stimulation with the equivalent antigens.
Results from one representative donor are shown in Panel D. Panel E
shows that the lines are polyfunctional as assessed using ICS for
IFN and TNF in one representative donor.
[0022] FIG. 6: Multivirus-specific CTLs can be expanded in vitro.
On day 9 after initial stimulation CTLs were restimulated using
pepmix-pulsed PHA blasts. Panel A shows the expansion of CTLs from
initiation (day 0) to day 16, following a 2nd stimulation on day
9/10 (n=4). CTL expansion was evaluated using trypan blue exclusion
and results are shown as mean cell numbers+/-STDEV. Panel B shows
results from 1 representative donor illustrating the antigen
specificity of CD3/CD8+ and CD3/CD8- (CD4+) CTLs after the 2nd
round of stimulation using IFN ICS. Panel C shows summary results
from 6 donors after the 1st (day 9) and 2nd (day 16) stimulation,
using IFN ELIspot as a readout. Results are expressed as
SFC/1.times.10.sup.5 input cells +/-STDEV and the control was IFN
release in response to stimulation with irrelevant pepmix. The
cytotoxic abilities of the generated CTLs were evaluated by
standard 4-6 hr Cr.sup.51 release assay using pepmix-pulsed PHA
blasts as targets. Specific lysis after the 1st and 2nd stimulation
from 2 representative donors are shown in Panel D.
[0023] FIG. 7: Phenotype and specificity of Penton and
LMP2-specific CTLs generated in the presence of different
growth-promoting cytokines. Phenotypic analyses of CTLs on day 9
after initiation with Penton (upper left) or LMP2 (upper right
panel) pepmixes and culture in the presence or absence of different
cytokines. Results are presented as mean % positive cells+/-STDEV.
CTLs of 3 donors were tested for specificity by IFN ELIspot.
Results are expressed as SFC/1.times.10.sup.5 input cells+/-STDEV
and the control was IFN release in response to stimulation with an
irrelevant pepmix.
[0024] FIG. 8: CD3+ T cell expansion after addition of
growth-promoting cytokines. Total T cell numbers were calculated
based on total cell numbers evaluated by cell counting using trypan
blue exclusion and the percentage of CD3+ T cells detected on day 9
after CTL initiation assessed by flow cytometric analysis. Results
from 5 donors are shown (mean cell numbers+/-SEM).
[0025] FIG. 9: TCR avidity is comparable in Hexon DC
plasmid-activated and pepmix-stimulated PBMCs. TCR avidity of
Hexon-specific CTLs stimulated with plasmid nucleofected DCs or
pepmix stimulated PBMCs was tested by serial dilution of Hexon
pepmix or HLA-A1 restricted peptide TDL with IFN ELIspot as
readout. Results are plotted as % of maximum SFC.
[0026] FIG. 10: Comparable expansion of CTLs stimulated with pooled
vs. single pepmixes. Cell expansion of CTL generated from 2 donors
was evaluated using trypan blue exclusion 9 days after PBMC
stimulation. Results are expressed as mean cell
numbers+/-STDEV.
[0027] FIG. 11: Lack of alloreactivity in pepmix-stimulated PBMCs.
The alloreactive potential of pepmix-activated CTL stimulatd either
once (n=4) or twice (n=2) was tested by Cr.sup.51 release assay
against a range of allogeneic HLA-mismatched PHA blasts as
targets.
DETAILED DESCRIPTION OF THE INVENTION
I. Definitions
[0028] In keeping with long-standing patent law convention, the
words "a" and "an" when used in the present specification in
concert with the word comprising, including the claims, denote "one
or more." Some embodiments of the invention may consist of or
consist essentially of one or more elements, method steps, and/or
methods of the invention. It is contemplated that any method or
composition described herein can be implemented with respect to any
other method or composition described herein.
[0029] The term "tumor antigen" as used herein refers to an
antigenic substance produced/expressed on tumor cells and which
triggers an immune response in the host.
[0030] The term "viral antigen" as used herein refers to an antigen
that is protein in nature and is closely associated with the virus
particle. In specific embodiments, a viral antigen is a coat
proteins.
II. General Embodiments of the Invention
[0031] In certain aspects of the invention, the present invention
concerns the development of CTLs that target one or more antigens
from at least one virus or at least one tumor antigen, for example.
In some cases, the CTLs target one or more antigens from two or
more viruses or two or more tumors.
[0032] The present invention concerns methods for generating CTL
lines with specificity against multiple tumor antigens or multiple
viruses in at least general embodiments. In methods of producing
CTLs the antigen is presented to PBMCs (for example) in the form of
one or more peptides that span some or all of the antigen. The
antigenic peptides may be provided to the PBMCs in a library of
peptide mixtures, which may be referred to as pepmixes, and
multiple libraries of pepmixes may be provided to the same
collection of PBMCs. In some embodiments, the collection includes
both immunodominant and subdominant antigens.
[0033] In some embodiments, the present invention is utilized in
individuals after hematopoietic stem cell transplantation (HSCT)
Severe and fatal viral infections remain common after HSCT.
Adoptive transfer of cytotoxic T lymphocytes (CTLs) specific for
EBV, CMV and Adenoviral antigens can treat infections that are
impervious to conventional therapies, but broader implementation
and extension to additional viruses are limited by competition
between virus-derived antigens and time-consuming and laborious
manufacturing procedures. The invention provides a system that
rapidly generates a single preparation of polyclonal (CD4+ and
CD8+) CTLs that is consistently specific for 15 immunodominant and
subdominant antigens derived from 7 viruses (EBV, CMV, Adv, BK,
HHV6, RSV and Influenza) that commonly cause post-transplant
morbidity and mortality. CTLs can be rapidly produced (10 days) by
a single stimulation of donor PBMCs with a peptide mixture spanning
the target antigens in the presence of the potent pro-survival
cytokines IL4 and IL7. This approach reduces the impact of
antigenic competition with a consequent increase in the antigenic
repertoire and frequency of virus-specific T cells. The present
invention can be readily introduced into clinical practice and is a
cost-effective alternative to common anti-viral prophylactic agents
for allogeneic HSCT recipients.
III. Pathogens and Pathogenic Antigens
[0034] In some embodiments of the invention, the generated CTLs are
provided to an individual that has or is at risk of having a
pathogenic infection, including a viral, bacterial, or fungal
infection. The individual may or may not have a deficient immune
system. In some cases, the individual has a viral, bacterial, or
fungal infection following organ or stem cell transplant (including
hematopoietic stem cell transplantation), or has cancer or has been
subjected to cancer treatment, for example. In some cases the
individual has infection following an acquired immune system
deficiency.
[0035] The infection in the individual may be of any kind, but in
specific embodiments the infection is the result of one or more
viruses. The pathogenic virus may be of any kind, but in specific
embodiments it is from one of the following families: Adenoviridae,
Picornaviridae, Herpesviridae, Hepadnaviridae, Flaviviridae,
Retroviridae, Orthomyxoviridae, Paramyxoviridae, Papovaviridae,
Polyomavirus, Rhabdoviridae, or Togaviridae. In some embodiments,
the virus produces antigens that are immunodominant or subdominant
or produces both kinds. In specific cases, the virus is selected
from the group consisting of EBV, CMV, Adenovirus, BK virus, HHV6,
RSV, Influenza, Parainfluenza, Bocavirus, Coronavirus, LCMV, Mumps,
Measles, Metapneumovirus, Parvovirus B, Rotavirus, West Nile Virus,
Spanish influenza, and a combination thereof.
[0036] In some aspects the infection is the result of a pathogenic
bacteria, and the present invention is applicable to any type of
pathogenic bacteria. Exemplary pathogenic bacteria include at least
Mycobacterium tuberculosis, Mycobacterium leprae, Clostridium
botulinum, Bacillus anthracis, Yersinia pestis, Rickettsia
prowazekii, Streptococcus, Pseudomonas, Shigella, Campylobacter,
and Salmonella.
[0037] In some aspects the infection is the result of a pathogenic
fungus, and the present invention is applicable to any type of
pathogenic fungus. Exemplary pathogenic fungi include at least
Candida, Aspergillus, Cryptococcus, Histoplasma, Pneumocystis, or
Stachybotrys.
IV. Tumor Antigens
[0038] In embodiments wherein multiTAA-specific CTL are employed
for the treatment and/or prevention of cancer, a variety of TAA may
be targeted. Tumor antigens are substances produced in tumor cells
that trigger an immune response in a host.
[0039] Exemplary tumor antigens include at least the following:
carcinoembryonic antigen (CEA) for bowel cancers; CA-125 for
ovarian cancer; MUC-1 or epithelial tumor antigen (ETA) or CA15-3
for breast cancer; tyrosinase or melanoma-associated antigen (MAGE)
for malignant melanoma; and abnormal products of ras, p53 for a
variety of types of tumors; alphafetoprotein for hepatoma, ovarian,
or testicular cancer; beta subunit of hCG for men with testicular
cancer; prostate specific antigen for prostate cancer; beta 2
microglobulin for multiple myelom and in some lymphomas; CA19-9 for
colorectal, bile duct, and pancreatic cancer; chromogranin A for
lung and prostate cancer; TA90 for melanoma, soft tissue sarcomas,
and breast, colon, and lung cancer. Examples of tumor antigens are
known in the art, for example in Cheever et al., 2009, which is
incorporated by reference herein in its entirety.
[0040] Specific examples of tumor antigens include at least CEA,
MHC, CTLA-4, gp 100, mesothelin, PD-L1, TRP 1, CD40, EGFP, Her2,
TCR alpha, trp2, TCR, MUC 1, cdr2, ras, 4-1BB, CT26, GITR, OX40,
TGF-.alpha.. WT1, MUC1, LMP2, HPV E6 E7, EGFRvIII, HER-2/neu, MAGE
A3, p53 nonmutant, NY-ESO-1, PSMA, GD2, Melan A/MART1, Ras mutant,
gp 100, p53 mutant, Proteinase3 (PR1), bcr-abl, Tyrosinase,
Survivin, PSA, hTERT, EphA2, PAP, ML-IAP, AFP, EpCAM, ERG (TMPRSS2
ETS fusion gene), NA17, PAX3, ALK, Androgen receptor, Cyclin B1,
Polysialic acid, MYCN, RhoC, TRP-2, GD3, Fucosyl GM1, Mesothelin,
PSCA, MAGE A1, sLe(a), CYP1B1, PLAC1, GM3, BORIS, Tn, GloboH,
ETV6-AML, NY-BR-1, RGS5, SART3, STn, Carbonic anhydrase IX, PAX5,
OY-TES1, Sperm protein 17, LCK, HMWMAA, AKAP-4, SSX2, XAGE 1, B7H3,
Legumain, Tie 2, Page4, VEGFR2, MAD-CT-1, FAP, PDGFR-.beta.,
MAD-CT-2, and Fos-related antigen 1, for example.
V. Generation of Pepmix Libraries
[0041] In some embodiments of the invention, a library of peptides
is provided to PBMCs ultimately to generate CTLs. The library in
particular cases comprises a mixture of peptides ("pepmixes") that
span part or all of the same antigen. Pepmixes utilized in the
invention may be from commercially available peptide libraries made
up of peptides that are 15 amino acids long and overlapping one
another by 11 amino acids, in certain aspects. In some cases, they
may be generated synthetically. Examples include those from JPT
Technologies (Springfield, Va.) or Miltenyi Biotec (Auburn,
Calif.). In particular embodiments, the peptides are at least 7, 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,
26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 or more amino acids in
length, for example, and in specific embodiments there is overlap
of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or
34 amino acids in length, for example. The mixture of different
peptides may include any ratio of the different peptides, although
in some embodiments each particular peptide is present at
substantially the same numbers in the mixture as another particular
peptide.
VI. Combination Therapy
[0042] In certain embodiments of the invention that concern CTLs
generated against tumor antigens, methods of the present invention
for clinical aspects are combined with other agents effective in
the treatment of hyperproliferative disease, such as anti-cancer
agents. An "anti-cancer" agent is capable of negatively affecting
cancer in a subject, for example, by killing cancer cells, inducing
apoptosis in cancer cells, reducing the growth rate of cancer
cells, reducing the incidence or number of metastases, reducing
tumor size, inhibiting tumor growth, reducing the blood supply to a
tumor or cancer cells, promoting an immune response against cancer
cells or a tumor, preventing or inhibiting the progression of
cancer, or increasing the lifespan of a subject with cancer. More
generally, these other compositions would be provided in a combined
amount effective to kill or inhibit proliferation of the cell. This
process may involve contacting the cancer cells with the expression
construct and the agent(s) or multiple factor(s) at the same time.
This may be achieved by contacting the cell with a single
composition or pharmacological formulation that includes both
agents, or by contacting the cell with two distinct compositions or
formulations, at the same time, wherein one composition includes
the expression construct and the other includes the second
agent(s).
[0043] Tumor cell resistance to chemotherapy and radiotherapy
agents represents a major problem in clinical oncology. One goal of
current cancer research is to find ways to improve the efficacy of
chemo- and radiotherapy by combining it with gene therapy. For
example, the herpes simplex-thymidine kinase (HS-tK) gene, when
delivered to brain tumors by a retroviral vector system,
successfully induced susceptibility to the antiviral agent
ganciclovir.sup.51. In the context of the present invention, it is
contemplated that cell therapy could be used similarly in
conjunction with chemotherapeutic, radiotherapeutic, or
immunotherapeutic intervention, in addition to other pro-apoptotic
or cell cycle regulating agents.
[0044] Alternatively, the present inventive therapy may precede or
follow the other agent treatment by intervals ranging from minutes
to weeks. In embodiments where the other agent and present
invention are applied separately to the individual, one would
generally ensure that a significant period of time did not expire
between the time of each delivery, such that the agent and
inventive therapy would still be able to exert an advantageously
combined effect on the cell. In such instances, it is contemplated
that one may contact the cell with both modalities within about
12-24 h of each other and, more preferably, within about 6-12 h of
each other. In some situations, it may be desirable to extend the
time period for treatment significantly, however, where several d
(2, 3, 4, 5, 6 or 7) to several wk (1, 2, 3, 4, 5, 6, 7 or 8) lapse
between the respective administrations.
[0045] Various combinations may be employed, present invention is
"A" and the secondary agent, such as radio- or chemotherapy, is
"B":
TABLE-US-00001 A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B
B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B
A/A/A/B B/A/A/A A/B/A/A A/A/B/A
[0046] It is expected that the treatment cycles would be repeated
as necessary. It also is contemplated that various standard
therapies, as well as surgical intervention, may be applied in
combination with the inventive cell therapy.
[0047] A. Chemotherapy
[0048] Cancer therapies also include a variety of combination
therapies with both chemical and radiation based treatments.
Combination chemotherapies include, for example, abraxane,
altretamine, docetaxel, herceptin, methotrexate, novantrone,
zoladex, cisplatin (CDDP), carboplatin, procarbazine,
mechlorethamine, cyclophosphamide, camptothecin, ifosfamide,
melphalan, chlorambucil, busulfan, nitrosurea, dactinomycin,
daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin,
etoposide (VP16), tamoxifen, raloxifene, estrogen receptor binding
agents, taxol, gemcitabien, navelbine, farnesyl-protein tansferase
inhibitors, transplatinum, 5-fluorouracil, vincristin, vinblastin
and methotrexate, or any analog or derivative variant of the
foregoing.
[0049] B. Radiotherapy
[0050] Other factors that cause DNA damage and have been used
extensively include what are commonly known as .gamma.-rays,
X-rays, and/or the directed delivery of radioisotopes to tumor
cells. Other forms of DNA damaging factors are also contemplated
such as microwaves and UV-irradiation. It is most likely that all
of these factors effect a broad range of damage on DNA, on the
precursors of DNA, on the replication and repair of DNA, and on the
assembly and maintenance of chromosomes. Dosage ranges for X-rays
range from daily doses of 50 to 200 roentgens for prolonged periods
of time (3 to 4 wk), to single doses of 2000 to 6000 roentgens.
Dosage ranges for radioisotopes vary widely, and depend on the
half-life of the isotope, the strength and type of radiation
emitted, and the uptake by the neoplastic cells.
[0051] The terms "contacted" and "exposed," when applied to a cell,
are used herein to describe the process by which a therapeutic
construct and a chemotherapeutic or radiotherapeutic agent are
delivered to a target cell or are placed in direct juxtaposition
with the target cell. To achieve cell killing or stasis, both
agents are delivered to a cell in a combined amount effective to
kill the cell or prevent it from dividing.
[0052] C. Immunotherapy
[0053] Immunotherapeutics, generally, rely on the use of immune
effector cells and molecules to target and destroy cancer cells.
The immune effector may be, for example, an antibody specific for
some marker on the surface of a tumor cell. The antibody alone may
serve as an effector of therapy or it may recruit other cells to
actually effect cell killing. The antibody also may be conjugated
to a drug or toxin (chemotherapeutic, radionuclide, ricin A chain,
cholera toxin, pertussis toxin, etc.) and serve merely as a
targeting agent. Alternatively, the effector may be a lymphocyte
carrying a surface molecule that interacts, either directly or
indirectly, with a tumor cell target. Various effector cells
include cytotoxic T cells and NK cells.
[0054] Immunotherapy, thus, could be used as part of a combined
therapy, in conjunction with the present cell therapy. The general
approach for combined therapy is discussed below. Generally, the
tumor cell must bear some marker that is amenable to targeting,
i.e., is not present on the majority of other cells. Many tumor
markers exist and any of these may be suitable for targeting in the
context of the present invention. Common tumor markers include
carcinoembryonic antigen, prostate specific antigen, urinary tumor
associated antigen, fetal antigen, tyrosinase (p97), gp68, TAG-72,
HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, estrogen receptor,
laminin receptor, erb B and p155.
[0055] D. Genes
[0056] In yet another embodiment, the secondary treatment is a gene
therapy in which a therapeutic polynucleotide is administered
before, after, or at the same time as the present invention
clinical embodiments. A variety of expression products are
encompassed within the invention, including inducers of cellular
proliferation, inhibitors of cellular proliferation, or regulators
of programmed cell death.
[0057] E. Surgery
[0058] Approximately 60% of persons with cancer will undergo
surgery of some type, which includes preventative, diagnostic or
staging, curative and palliative surgery. Curative surgery is a
cancer treatment that may be used in conjunction with other
therapies, such as the treatment of the present invention,
chemotherapy, radiotherapy, hormonal therapy, gene therapy,
immunotherapy and/or alternative therapies.
[0059] Curative surgery includes resection in which all or part of
cancerous tissue is physically removed, excised, and/or destroyed.
Tumor resection refers to physical removal of at least part of a
tumor. In addition to tumor resection, treatment by surgery
includes laser surgery, cryosurgery, electrosurgery, and
miscopically controlled surgery (Mohs' surgery). It is further
contemplated that the present invention may be used in conjunction
with removal of superficial cancers, precancers, or incidental
amounts of normal tissue.
[0060] Upon excision of part of all of cancerous cells, tissue, or
tumor, a cavity may be formed in the body. Treatment may be
accomplished by perfusion, direct injection or local application of
the area with an additional anti-cancer therapy. Such treatment may
be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or
every 1, 2, 3, 4, and 5 weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, or 12 months. These treatments may be of varying dosages as
well.
[0061] F. Other agents
[0062] It is contemplated that other agents may be used in
combination with the present invention to improve the therapeutic
efficacy of treatment. These additional agents include
immunomodulatory agents, agents that affect the upregulation of
cell surface receptors and GAP junctions, cytostatic and
differentiation agents, inhibitors of cell adhesion, or agents that
increase the sensitivity of the hyperproliferative cells to
apoptotic inducers. Immunomodulatory agents include tumor necrosis
factor; interferon alpha, beta, and gamma; IL-2 and other
cytokines; F42K and other cytokine analogs; or MIP-1, MIP-1beta,
MCP-1, RANTES, and other chemokines. It is further contemplated
that the upregulation of cell surface receptors or their ligands
such as Fas/Fas ligand, DR4 or DR5/TRAIL would potentiate the
apoptotic inducing abililties of the present invention by
establishment of an autocrine or paracrine effect on
hyperproliferative cells. Increases intercellular signaling by
elevating the number of GAP junctions would increase the
anti-hyperproliferative effects on the neighboring
hyperproliferative cell population. In other embodiments,
cytostatic or differentiation agents can be used in combination
with the present invention to improve the anti-hyerproliferative
efficacy of the treatments. Inhibitors of cell adhesion are
contemplated to improve the efficacy of the present invention.
Examples of cell adhesion inhibitors are focal adhesion kinase
(FAKs) inhibitors and Lovastatin. It is further contemplated that
other agents that increase the sensitivity of a hyperproliferative
cell to apoptosis, such as the antibody c225, could be used in
combination with the present invention to improve the treatment
efficacy.
[0063] Hormonal therapy may also be used in conjunction with the
present invention or in combination with any other cancer therapy
previously described. The use of hormones may be employed in the
treatment of certain cancers such as breast, prostate, ovarian, or
cervical cancer to lower the level or block the effects of certain
hormones such as testosterone or estrogen. This treatment is often
used in combination with at least one other cancer therapy as a
treatment option or to reduce the risk of metastases.
[0064] DNA methyltransferase inhibitors and/or histone deacetylase
inhibitors. Exemplary DNA methyltransferase inhibitors include, for
example, 5-azacytidine, 5-aza-2'-deoxycytidine,
1-beta-D-arabinofuranosyl-5-azacytosine and dihydro-5-azacytidine.
Exemplary HDAC inhibitors include hydroxamic acids, such as
trichostatin A; cyclic tetrapeptides (such as trapoxin B), and the
depsipeptides; benzamides; electrophilic ketones; and the aliphatic
acid compounds such as phenylbutyrate and valproic acid.
VII. Kits of the Invention
[0065] Any of the compositions described herein may be comprised in
a kit. In a non-limiting example, a library of pepmixes may be
comprised in a kit, any type of cells may be provided in the kit,
and/or reagents for manipulation of pepmixes and/or cells may be
provided in the kit. The components are provided in suitable
container means.
[0066] The kits may comprise a suitably aliquoted compositions of
the present invention. The components of the kits may be packaged
either in aqueous media or in lyophilized form. The container means
of the kits will generally include at least one vial, test tube,
flask, bottle, syringe or other container means, into which a
component may be placed, and preferably, suitably aliquoted. Where
there are more than one component in the kit, the kit also will
generally contain a second, third or other additional container
into which the additional components may be separately placed.
However, various combinations of components may be comprised in a
vial. The kits of the present invention also will typically include
a means for containing the components in close confinement for
commercial sale. Such containers may include injection or
blow-molded plastic containers into which the desired vials are
retained.
[0067] However, the components of the kit may be provided as dried
powder(s). When reagents and/or components are provided as a dry
powder, the powder can be reconstituted by the addition of a
suitable solvent. It is envisioned that the solvent may also be
provided in another container means.
EXAMPLES
[0068] The following examples are presented in order to more fully
illustrate the preferred embodiments of the invention. They should
in no way, however, be construed as limiting the broad scope of the
invention.
Example 1
IL2, IL15 and IL4+7 Promote the Expansion of Peptide-Activated T
Cells In Vitro
[0069] To increase the range of viral antigens that could be
recognized by a single CTL line and to mitigate the impact of
antigenic competition in order to retain both high and low
frequency T cells, the inventors stimulated PBMCs in the presence
of different Th1, pro-proliferative and pro-survival cytokines. The
inventors then compared the frequency and repertoire of responding
cells to those generated by conventional activation in the absence
of cytokines. In exploratory experiments PBMCs were simulated with
a pepmix (peptide library of overlapping 15mers) spanning the
immunodominant CMV-pp65 antigen, then expanded without cytokines,
or with media supplemented with (i) IL15 (5 ng/ml), (ii) IL2 (20
U/ml), or (iii) IL4 (1666 U/ml)+IL7 (10 ng/ml). After 9-12 days the
inventors assessed cell expansion, phenotype, specificity and
function.
[0070] Cultures supplemented with IL15 or IL4+7 showed the greatest
overall expansion (5.+-.0.6 and 3.7.+-.0.5 fold increase,
respectively) over 9 days (n=5). Cultures that were stimulated in
the absence of cytokines did not expand (0.6.+-.0.04), while the
IL2 condition was intermediate (2.7.+-.0.1) (FIG. 1A). To determine
whether the superior cell numbers were a consequence of improved T
cell proliferation, enhanced survival, or the combination, cells
were labeled with CFSE on day 0 and then analyzed every 2-3 days to
measure cell doubling, while live and apoptotic/necrotic cells were
distinguished by Annexin-PI staining (not shown). Flow cytometric
analysis demonstrated no difference in the number of cell divisions
from day 0-5. However, from day 5 onward cells cultured in
cytokines continued to divide, whereas in their absence, cell
division was reduced and viability was consistently lower (FIG.
1B). These data indicate that the improved survival of
proliferating cells made the primary contribution to the observed
increase in cell numbers in cytokine-supplemented cultures.
Example 2
IL4+7 Support the Selective Expansion of Polyclonal, TH1-Polarized
T Cells
[0071] Optimal in vivo T cell persistence and activity requires
both helper (CD4+) and cytotoxic (CD8+) T cells.sup.23. The
inventors therefore used phenotypic analyses to determine that the
cells in the cytokine-supplemented cultures reflected the selective
expansion of polyclonal T cells. The inventors found the lowest
frequency of CD3+ T cells in cultures supplemented with IL2 or IL15
(72.8.+-.2.1% and 61.3.+-.3.7%, respectively), which instead
contained significantly higher numbers of CD56+ NK cells than other
conditions (27.1.+-.2.3% and 37.7.+-.3.7%, respectively) (n=5). By
contrast, IL4+7 cultures were comprised almost entirely of CD3+ T
cells (92.6.+-.0.4%), with both CD8+ T cells and significantly more
CD4+ T cells (61.+-.2.7%) than the other cytokine-supplemented
conditions (IL2 26.+-.4%, IL15 17.6.+-.4.3%, p=0.024, p=0.004,
respectively) (FIG. 1C). To confirm that both CD8+ and CD4+ T cells
were antigen-specific and produced effector cytokines the inventors
performed intracellular cytokine staining (ICS) for IFN. FIG. 1D
shows representative results from 1 donor, while FIG. 1E shows
summary results for 3 donors. The data confirm that
IL4+7-supplemented cultures contained antigen-specific
IFN-producing T cells in both compartments (CD4+ 39.3%.+-.16.4%,
CD8+ 22.2%.+-.2.2%), at levels substantially higher than in other
conditions (no cytokine: CD4+ 2.3%.+-.3.9%, CD8+ 0.8%.+-.0.5%;
IL15: CD4+ 1.7%.+-.1.4%, CD8+ 13.9%.+-.2.8% and IL2: CD4+
2.2%.+-.3.1%, CD8+ 12.6%.+-.2.6%, n=3). Similar results were
obtained using pepmixes from subdominant Adv (Penton) and EBV
(LMP2) viral antigens; indeed outgrowth of NK cells was even more
evident in the IL2 and IL15-supplemented conditions (FIG. 7).
[0072] IL4 is a prototypic Th2 cytokine, therefore to more
comprehensively evaluate the cytokine profile of the induced CTLs
the supernatant of antigen-activated T cells was assessed using
luminex array. FIG. 1F shows that, in addition to IFN.gamma., the
IL4+7-supplemented lines produced the prototypic Th1 cytokines
GM-CSF, IL-2 and TNF.alpha., at levels similar to that of
IL2-induced CTLs. In addition, levels of Th2 cytokines (IL5 and
IL13) were not substantially different and there was no evidence of
regulatory T cell outgrowth, as assessed by CD4/CD25/FoxP3+
staining (FIG. 1G). Thus, IL4, in combination with IL7, induces
selective expansion of polyclonal, Th1-polarized T cells that
produce multiple effector cytokines upon stimulation (FIG. 8).
Example 3
Overlapping 15mer Peptide Libraries Activate T Cells with Similar
Specificity and Avidity to Those Generated Using
Endogenously-Processed Full Length Antigen
[0073] To address concerns that pepmixes might reactivate low
avidity T cells unable to recognize antigens that are naturally
processed and presented by virus-infected cells, the inventors
compared pp65 pepmix-activated CTLs with those generated using DCs
nucleofected with a DNA plasmid encoding the same
antigen.sup.20,21. After activation, each set of cells was expanded
in IL4+7. Expansion was similar between the groups, with
107.+-.23.4.times.10.sup.6 cells generated using pepmix-pulsed
PBMCs (7.2 fold expansion) versus 130.3.+-.46.9.times.10.sup.6
cells in the DC-stimulated cultures (8.7 fold expansion) (FIG. 2A)
(n=3). Phenotypic analysis demonstrated that the pepmix-activated
CTLs were predominantly CD4+ (74.3.+-.19.3%), with a minor CD8+
component (22.8.+-.19.2%), as were the plasmid-activated CTLs (CD4+
70.6.+-.14.2% and CD8+ 26.5.+-.13.4%) and both expressed similar
levels of the memory and activation markers CD62L, CD28 and CD45RO
(61.+-.46.7%, 86.5.+-.3.5%, 92.+-.7.1% pepmix vs. 77.+-.28.3%,
85.5.+-.0.7%, 87.5.+-.13.4% plasmid) (FIG. 2B). The inventors next
compared the breadth of epitopes recognized by measuring responses
to 110 20mer peptides (overlapping by 15aa) spanning CMV-pp65 and
arranged into 22 pools such that each peptide was represented in 2
pools.sup.24. FIG. 2C shows that both the recognition of a given
peptide and the magnitude of the response thereto was little
changed by the antigen source. Finally, the inventors compared
functional avidity by IFN.gamma. ELIspot using log dilutions of the
pp65 pepmix or epitope peptides (A2-NLV and A24-QAD) as a stimulus.
As shown in FIG. 2D, there was no significant difference in the
avidity of the CTLs. This data was confirmed for other viral
antigens using Adv-Hexon pepmix and viral antigen-encoding plasmid
as a stimulus (FIG. 9).
Example 4
15mer Peptides Activate CD4+ and CD8+ T Cells as Efficiently as
Long (20mer or 30mer) Peptides
[0074] Since CD4+ epitopes (>20aa) may be longer that CD8
epitopes (8-10aa) the inventors next determined whether longer
peptides would induce higher frequencies of antigen-specific CD4+ T
cells. The inventors obtained three overlapping peptide libraries
(#1-15mers overlapping by 11, #2-20mers overlapping by 15, and
#3-30mers overlapping by 15) spanning the C terminus (aa539-953) of
Adv-Hexon; a region rich in both CD4+ and CD8+ epitopes.sup.25,26
(FIG. 3A). The inventors directly stimulated PBMCs with each of the
libraries and evaluated the phenotype, epitope specificity and
breadth of the lines.
[0075] Phenotypically the lines were comparable, with a
predominance of CD4+ cells (mean 56.+-.5.5% vs. 59.+-.5.8% vs.
60.+-.6%) and a minor CD8+ component (mean 21.+-.0.2% vs.
20.+-.0.1% vs. 16.+-.0.2%), and similar levels of the memory and
activation markers CD62L, CD28 and CD45RO (CD62L--60.+-.1.9% vs.
57.+-.1.9% vs. 51+/-1.6%, CD28--88.+-.0.6% vs. 84.+-.2.1%, vs.
89.+-.0.6% and CD45RO--58.+-.1.7% vs. 60.+-.1.6% vs. 60.+-.1.2%)
(15mer vs. 20mer vs. 30mer) (n=6). To learn whether the spectrum of
epitopes recognized differed based on the stimulating library; the
inventors rechallenged the induced CTLs with subpools of peptides
from each library and found no consistent or statistically
significant differences in the breadth of peptides recognized.
Results for the 15mer minipool rechallenge are shown in FIG. 3C.
Since 15mer pepmixes are readily available as both research and
clinical products the inventors performed all subsequent
experiments with this antigen source.
Example 5
Generation of a Single T Cell Culture with Simultaneous Specificity
for Adv, Ebv and Cmv
[0076] After successfully generating CTLs using peptides derived
from a single viral antigen and culture in IL4+7, the inventors
next prepared a single culture of CTLs simultaneously recognizing
CMV, EBV, and Adv. For each virus the inventors targeted
immunogenic antigens; CMV-IE1 and pp65, Adv-Hexon and Penton, and
EBV-EBNA1, LMP2 and BZLF1.sup.8,9,17,18,27-31 and pulsed PBMCs with
the relevant pepmixes before culture in IL4+7. After 9-12 days the
inventors compared the antiviral reactivity of the resulting CTLs
with those generated using our current clinical trivirus CTL
protocol which uses DCs nucleofected with plasmids encoding the
same antigens as a stimulus.sup.20,21 (FIG. 4). IFN.gamma. ELIspot
confirmed that pepmix-generated CTLs from 4 donors had antiviral
activity against all three viruses and seven stimulating antigens.
The frequency of T cells reactive against EBV (EBNA1, LMP2, BZLF1)
and CMV (IE1, pp65) was comparable irrespective of the stimulus. In
contrast, all 4 donors had significantly more Adv-reactive T cells
(Hexon and Penton) in pepmix-stimulated cultures [Hexon--median
462.3, range 373-572.5 vs. median 112, range 53-421.5
SFC/2.times.105 CTL; p=0.01, Penton--median 317, range 105.5-345
vs. median 51.25, range 4-134 SFC/2.times.105 CTL, p=0.02, pepmix
vs. plasmid, respectively].
Example 6
Extension to Additional Viruses
[0077] To determine whether the direct pepmix stimulation approach
could be extended to generate multivirus-specific CTL lines
targeting a broader spectrum of different clinically relevant
viruses the inventors stimulated PBMCs with pepmixes spanning 2 or
3 T cell immunogenic antigens from CMV, Adv, EBV, BK, Influenza,
RSV and HHV6 (Table 1).
TABLE-US-00002 TABLE 1 Exemplary Antigens from Exemplary Viruses
Virus Antigen EBV EBNA-1, LMP2, BZLF1 CMV IE-1, pp65 Adenovirus
Hexon, Penton BK virus LT, VP-1 Influenza MP1, NP1 RSV N, F HHV-6
U14, U90
[0078] To determine whether antigenic competition would preclude
pooling the inventors segregated the pepmixes and stimulated PBMCs
with minipools containing pepmixes from, A) each virus; B)
immunodominant (CMV, RSV, Flu, HHV6) and sub-dominant (Adv, EBV,
BK) viruses; C) lytic (Adv, RSV, Flu) and latent (EBV, CMV, HHV6,
BK) viruses, or D) a mastermix of all pepmixes (FIG. 5A). There was
no difference in either the rate of expansion (FIG. 10), the
overall specificity or magnitude of the response directed against
each antigen, irrespective of the composition of the stimulating
pepmix pool (FIG. 5B). Thus, all further studies used the mastermix
(condition D). FIG. 5C shows 8 additional CTL lines with consistent
multivirus specificity. The highest responses were seen against
CMV-pp65 and Adv-Hexon (951.6.+-.82.1 and 461.4.+-.19.2
SFC/1.times.10.sup.5 CTL) while activity against HHV6-U90,
EBV-BZLF1 and EBV-LMP2 was weakest (26.9.+-.4.2, 35.6.+-.5,
39.6.+-.2.6 SFC/1.times.10.sup.5 CTL). Adv-Penton, Influenza-MP1
and RSV-F demonstrated intermediate response rates (191.+-.13.7,
117.6.+-.8.6, 90.1.+-.10.3 SFC/1.times.10.sup.5 CTL, respectively)
(FIG. 5C). The lines were polyclonal and polyfunctional with
activity against the stimulating viruses detectable in both CD4+
and CD8+ fractions (FIG. 5D), and reactive cells produced both
IFN.gamma. and TNF.alpha. superior in vivo activity.sup.32,33. FIG.
5E shows the results for one representative donor in whom 63% of
all Adv, 55% of CMV, 40% of EBV, 46% of RSV, 36% of Influenza and
28% of HHV6-specific CTLs produced both IFN.gamma. and TNF.alpha.
after antigenic stimulation. ICS for IFN.gamma. and/or TNF.alpha.
showed that 67.7.+-.13.3% of all T cells in multivirus cultures
were antigen-specific. This percentage is likely an underestimate
since some virus-specific CTLs do not produce cytokines or produce
effector cytokines other than IFN.gamma. and TNF.alpha..sup.33.
Finally, even though these CTLs had received only a single
stimulation there was no evidence of alloreactivity, assessed by
Cr.sup.51 release assay using HLA-mismatched PHA blasts as targets
(FIG. 10), an important consideration if these cells are to be used
for the treatment of allogeneic HSCT recipients.
Example 7
Multivirus-Specific CTL can be Expanded In Vitro
[0079] To discover whether multivirus-specific CTLs could be
further expanded to provide numbers suited for third party or
"off-the-shelf" use, the inventors restimulated the cells with
autologous PHA blasts pulsed with the same mastermix of pepmixes.
Secondary expansion of a mean of 8.4.+-.2 fold was obtained over 7
days, to a final cell number of 604.6.+-.23.7.times.106 (FIG. 6A).
FIG. 6B shows that the expanded CTLs remained polyclonal, with
activity detected in both CD4+ and CD8+ compartments. Expansion was
associated with an overall increase in the magnitude of the
response directed against all of the stimulating antigens on day 16
relative to day 9 (FIG. 6C) so that >80% of cells in the
restimulated cultures produced IFN.gamma. and/or TNF.alpha.
Similarly, these expanded cultures had greater cytolytic activity,
ranging from >60% (CMV) to 14% (BK), demonstrating retained
specificity for both sub-dominant and immunodominant
antigens/viruses without alloreactivity (FIG. 11).
Example 8
Significance of Certain Embodiments of the Invention
[0080] The inventors have shown that in at least some embodiments
they can rapidly generate polyclonal, CD4+ and CD8+ T cells with
specificities directed to a wide range of lytic and latent viruses
responsible for infection in the immunocompromised host and after
HSCT. These cells were Th1-polarized, had high avidity for a
multiplicity of individual viral antigens, produced multiple
effector cytokines upon stimulation, and killed virus-infected
targets without alloreactivity. Because the inventors generated
these T cells using combinations of clinically-available
peptide-libraries and pro-survival cytokines, our approach should
be well suited to clinical application.
[0081] While CMV, EBV and Adv are the most frequently detected
viral infections following allogeneic HSCT, recipients are also
susceptible to numerous other viruses, including BK, JC, HHV6,
HHV7, influenza, parainfluenza, coronavirus, and RSV, all of which
may cause severe morbidity and mortality.sup.1,2. Several of these
viruses are only seasonally detected (e.g. influenza, RSV) while
others, such as HHV7, JC, and coronavirus, are infrequent, so that
it is impracticable to cover all these pathogens post-transplant by
generating individualized patient and single virus-specific T cell
products. Hence, the inventors sought to develop a strategy that
would enable the production of a single CTL line with simultaneous
specificity for a multiplicity of antigens.
[0082] In the current clinical trials of virus-specific T cells,
the inventors have used EBV-LCL, adenovectors and/or viral
antigen-encoding DNA plasmids to generate virus-directed T
cells.sup.7-9,20,21. The use of full-length antigen ensures that
CTL can be generated from all donors, irrespective of HLA, and that
the antigen is physiologically processed by APCs and produces CTLs
that recognize multiple CD4+ and CD8+ T cell epitopes and have
sufficient avidity to kill virus-infected targets. The induction of
lines that recognize multiple epitopes also minimizes virus escape
due to epitope loss and produces potent and sustained anti-viral
activity in vivo.sup.34. However, the requirements for live
virus/vectors are barriers to broader and late phase clinical
studies, and also limit the number of pathogens to which a single T
cell line can be directed.sup.8,9. The inventors therefore
evaluated whether clinically applicable pepmixes could be used as
an alternative. Though clinical studies using minimal epitope
peptides as vaccines have resulted in immune tolerance or the
activation of low avidity T cells.sup.35, Melief and colleagues
recently demonstrated improved results with long (22-45aa) peptides
containing both CD4+ and CD8+ epitope sequences.sup.36. They
observed that these long peptides were processed endogenously,
presented to T cells by APCs, and induced both helper and cytotoxic
T cells, resulting in robust and effective CTL responses.sup.36.
Based on these data, the inventors chose to use a whole antigen
source in the form of overlapping peptide libraries, but for
optimal induction of polyclonal CTL the inventors compared peptides
of different lengths (15mers, 20mers and 30mers) for stimulation.
However, the inventors saw no difference in the phenotype,
specificity or epitope breadth of our lines, highlighting the
differences between delivering peptides as a vaccine, where one
relies on endogenous APCs to take up and process antigen versus in
vitro T cell activation using professional APCs within PBMCs at
optimal effector:target ratios.sup.37. Given the ready clinical
availability of pepmixes containing 15mer peptides that cover all
possible CD8+ and the majority of CD4+ epitopes, the inventors
substituted this antigen source and were able to demonstrate
equivalency to "conventionally generated" CTLs with respect to both
epitope specificity and avidity.sup.20,21.
[0083] The inventors next addressed how best to extend the breadth
of antigen/epitope specificities that could be accommodated within
a single CTL line. Physiologically, T cells are activated when they
receive signals from TCR stimulation (signal 1), co-stimulation
(signal 2), and cytokines (signal 3). The "conventional CTLs" are
activated in the absence of exogenous cytokines, a deficit that
appears to adversely affect their proliferative capacity in vitro
and also increases their susceptibility to activation induced cell
death (AICD), likely resulting in a more restricted repertoire of
epitope recognition. Consistent with this possibility, both the
frequency and breadth of cells with viral specificity could be
increased by supplementing cultures with inflammatory and
pro-survival cytokines at initiation. The inventors chose to test
cytokines that support cell proliferation in vitro and in vivo
(IL2, IL15).sup.38,39, as well as combinations (IL4+7) that also
support the retention of a central memory phenotype, and promote
the survival of activated T cells by upregulation of anti-apoptotic
molecules e.g. Bcl-2.sup.40-43. Only lines supplemented with IL4+7
selectively promoted the expansion and survival of both CD4+ and
CD8+ virus-specific T cells: of note, the induced cells were
Th1-polarized despite exposure to IL4, a prototypic Th2 cytokine.
Given the clinical availability of both cytokines and their safety
in human clinical trials.sup.44,45, IL4+7 fulfilled the
requirements of the current study, however other pro-inflammatory
cytokines capable of mimicking the milieu present during viral
infection may produce similar benefits. For example, von Rossum and
colleagues recently reported that CD3/28-activated CD8+ T cells
cultured in an inflammatory cocktail consisting of IL1+IL6+IL23
underwent significantly less cell death after activation as
compared with cells activated in any of the cytokines alone or
activated in the presence of IL12.sup.46.
[0084] The direct stimulation of PBMCs with pepmixes and culture in
cytokine-supplemented conditions also allowed us to overcome a
second major barrier to increasing the spectrum of viruses targeted
in a single CTL line, namely antigenic competition resulting from
the use of a common APC to simultaneously present multiple
antigenic components from different viruses.sup.8,9. Antigenic
competition results both from limited access of peptides to HLA
molecules and physical constraints on the simultaneous stimulation
of both high and low frequency T cells.sup.13,14. To overcome these
issues, investigators have used artificial APCs (AAPCs) that are
engineered with molecules to provide the necessary TCR and
co-stimulatory events required for immune synapse formation.sup.47.
However, to avoid the inevitable complexities and costs of
introducing a gene-modified cellular product into the manufacturing
process, the inventors evaluated whether patient PBMCs themselves
could act as both a source of antigen presenting and responding
cells. B cells, monocytes and macrophages may all have the capacity
to present antigen to T cells and these APCs can utilize endo- and
exopeptidases to liberate class I or class II epitopes from 15mer
peptides.sup.48,49. By taking advantage of these properties, the
inventors can avoid reliance on a single APC endogenously
expressing multiple antigens at different levels as a shared T cell
stimulator, and instead have a diverse group of APCs in which each
cell has the potential to display a diverse repertoire of peptides,
allowing sufficient access for both high and low frequency T cells.
Thus, antigenic competition both within the APC and between T cells
could be alleviated. As proof of principle, the inventors generated
a single culture of T cells with reactivity for 15 antigens derived
from 7 latent and lytic viruses (EBV, CMV, BK, HHV6, Adv, Flu, and
RSV) using pooled pepmixes as a stimulus and saw no evidence of
competition. Additional pathogens can be included in this platform,
although in some embodiments ultimately APC numbers can eventually
become limiting; thus additions must be performed in a stepwise
manner and one must evaluate changes in the frequency and breadth
of T cell recognition of all peptides in the mix.
[0085] Critically for clinical feasibility, the approach was able
to produce large numbers of virus-specific T cells. By seeding just
1.5.times.10.sup.7 PBMCs in the G-Rex and a single in vitro
stimulation the inventors could regularly manufacture
1.times.10.sup.8 CTLs within 10 days, with a >10-fold enrichment
in virus-specific cells and a corresponding reduction in
alloreactive T cells to levels observed in repetitively stimulated
conventional CTLs, which have a proven safety record in
vivo.sup.7-9,50. Thus, using our new manufacturing technology the
inventors predict that multivirus-specific CTL will be safe for
infusion after a single exposure to pepmixes and will provide broad
spectrum anti-viral protection without GvHD. Should additional
cells be required, for example if banked virus-specific CTLs are
established for 3rd party recipients, a second stimulation using
pepmix-pulsed PHA blasts can expand the total number of CTLs
without impairing their epitope specificity or breadth.
Example 8
Exemplary Materials and Methods
[0086] A. Donors and Cell Lines
[0087] PBMCs were obtained from healthy volunteers with informed
consent using a Baylor College of Medicine IRB-approved protocol.
PBMCs were used to generate DCs, CTL lines and PHA blasts. PHA
blasts were generated from PBMC (2.times.10.sup.6/ml) using PHA (5
.mu.g/ml) and maintained in CTL media (RPMI 1640, 45% Click's
(Irvine Scientific, Santa Ana, Calif.), 2 mM GlutaMAX TM-I, and 5%
Human AB Serum) supplemented with IL2 (100 U/ml, NIH, Bethesda,
Va.), which was replenished every 3 days.
[0088] B. CTL Generation--Peptide Stimulation
[0089] i. Peptides/Pepmixes
[0090] For PBMC stimulation the inventors used commercially
available pepmixes (15mers overlapping by 11aa spanning EBV-LMP2,
BZLF1, EBNA1; Adv-Penton, Hexon; CMV-pp65, IE-1; BKV-VP1, large T;
Influenza A-MP1 (H3N2), NP (H3N2); RSV-F, N, JPT Technology,
Berlin, Germany. Pepmixes spanning HHV6 U14 and U90 were
synthesized by Genemed Synthesis Inc., San Antonio, Tex. USA.
Peptide libraries spanning the 414aa C-terminus of Adv-Hexon were
synthesized by Proimmune, Oxford, UK or Alta Bioscience, University
of Birmingham, Edgbaston, Birmingham, UK. Lyophilized peptides were
reconstituted at 5 mg/ml in DMSO.
[0091] ii. PBMC Stimulation
[0092] 15.times.10.sup.6 fresh/frozen PBMCs were pelleted in a 15
ml tube and pulsed for 30-60 min at 37.degree. C. with peptide
libraries/pepmixes, either singly or pooled, at a concentration of
100 ng/peptide/15.times.10.sup.6 PBMCs. After incubation cells were
resuspended in CTL media alone or supplemented with cytokines (as
outlined below) and transferred to a G-Rex10 (Wilson Wolf
Manufacturing Corporation, New Brighton, Minn.)
(15.times.10.sup.6/G-Rex10) or plated out in a 24-well plate
(2.times.106/well). Media and cytokines were replenished on day 5,
and cultures were split when they reached a density
>50.times.10.sup.6/G-Rex10 or >3.times.10.sup.6 cell/24-well.
On day 9-12, CTLs were harvested, counted and used for phenotypic
and functional studies.
[0093] iii. Cytokines for Promoting CTL Activation and
Expansion
[0094] The inventors compared 4 conditions; (i) no cytokine, (ii)
IL7 (10 ng/ml)+IL4 (1666 U/ml), (iii) IL15 (5 ng/ml) (R&D
Systems, Minneapolis, Minn.) and (iv) IL2 (20 U/ml). Cytokines were
added to CTLs at day 0 and replenished on day 5. In some
embodiments, 400U of IL4 is employed.
[0095] iv. CTL Expansion
[0096] For expansion CTLs were restimulated at a S:R ratio of 1:1
with irradiated (30Gy) pepmix-pulsed autologous PHA blasts in CTL
media with IL4+7 and IL15 (5 ng/ml) on the day of restimulation and
fed with IL15 twice weekly. Seven days later CTLs were harvested,
and used for further studies.
[0097] C. Flow Cytometry
[0098] i. Immunophenotyping
[0099] CTLs were surface-stained with monoclonal antibodies to:
CD3, CD4, CD8, CD16, CD56, CD28, CD45RO, and CD62L (Becton
Dickinson BD, Franklin Lakes, N.J.). Cells were washed once with
phosphate-buffered saline (PBS) (Sigma, St Louis, Mo.) containing
2% FBS (HyClone, Thermo Fisher Scientific Inc, NH), pelleted, and
antibodies added in saturating amounts (10 .mu.l). After 15 min at
4.degree. C. in the dark, cells were washed twice and analyzed.
Approximately 20,000 live cells were acquired using a FACSCalibur
equipped with Cell Quest software
[0100] ii. CFSE
[0101] To measure cell proliferation PBMCs were isolated, pelleted
and pulsed with pp65 pepmix (100 ng/15.times.10.sup.6 PBMC) for
30-60 min. Next PBMCs were washed twice using PBS+0.1% FBS and
incubated for 10 min with 150 l/20.times.10.sup.6 PBMC 10 .mu.M
CSFE. Subsequently FBS was added at a 1:1 ratio and incubated for
10 min at 37 C.degree.. After CFSE labeling PBMCs were washed twice
using PBS+2% FBS and plated at a concentration of
1.times.10.sup.6/ml in CTL media with cytokines. Dilution of CFSE
was examined every 2-3 days by flow after surface staining with
CD3, CD4, CD8 and CD56.
[0102] iii. FoxP3 Staining
[0103] To measure regulatory T cells Foxp3 staining was performed
using the e-Bioscience FoxP3 staining kit. Briefly, CTLs were
rested in CTL media for 48 h, then 1.times.10.sup.6 CTLs were
resuspended in PBS+2% FBS and surface stained for CD3, CD25 and
CD4. After washing the cells were resuspended in 1 ml
Fixation/Permeabilizastion solution and incubated for 1 h at
4.degree. C., then washed, resuspended in permeabilization buffer
and incubated with 0.2 .mu.l isotype or 10 .mu.l FoxP3 antibody
(Clone PCH101) for 30 min at 4.degree. C. After a final wash cells
were acquired using a FACSCalibur equipped with Cell Quest
software.
[0104] iv. Intracellular Cytokine Staining
[0105] CTLs were harvested, resuspended at a concentration of
5.times.10.sup.6/ml in CTL media and plated at 200 .mu.l/well in a
96 well plate. The cells were then stimulated with 100 ng of test
or control pepmix in the presence of Brefeldin A (1 .mu.g/ml), (BD)
CD28 and CD49d (1 .mu.g/ml) for 5-7 hours. Subsequently, CTLs were
washed with PBS+2% FBS, pelleted, and surface stained with CD8, CD4
and CD3 (10 .mu.l/antibody/tube). After 15 mins, cells were washed
twice, pelleted, fixed and permeabilized with Cytofix/Cytoperm
solution (BD) for 20 mins at 4.degree. C. in the dark. After
washing twice with PBS/2% FBS containing 0.1% saponin (Calbiochem,
EMD Chemicals, NJ) cells were incubated with 20 .mu.l IFN.gamma.
and/or TNF.alpha. antibodies (BD) for 30 min at 4.degree. C. in the
dark. Cells were then washed twice with cold PBS/2% FBS containing
0.1% saponin and at least 200,000 live cells from each population
were analyzed with a FACSCalibur equipped with Cell Quest software
(BD).
[0106] D. Functional Studies
[0107] i. Multiplex Assay
[0108] To assess cytokine production the inventors used a multiplex
assays. 1.times.105 pp65-CTLs were restimulated using 500 ng/ml
pp65 or control pepmix. After 16 hrs supernatant was collected and
the cytokine profile assessed using the MILLIPLEX High Sensitivity
Human Cytokine Magnetic Bead Panel (Millipore, Billerica, Mass.).
Specifically, 50 .mu.l supernatant was incubated overnight at
4.degree. C. with cytokine antibody beads. After incubation,
samples were washed and incubated for 1 hr at room temperature (RT)
with the biotinylated detection antibody. Finally
Streptavidin-Phycoerythrin was added for 30 min at RT, then samples
were washed and analyzed using the Luminex 200 instrument. Samples
were run in duplicate.
[0109] Ii. Enzyme-Linked Immunospot Assay
[0110] The inventors used ELISpot to quantify IFN.gamma.-producing
T cells and assess the breadth of reactivity in the CTL lines. The
populations were serially diluted from 4-1.times.10.sup.5
cells/well, and antigen-specific activity measured after direct
pepmix or peptide mini-pool stimulation. Each condition was run in
triplicate. After 20 hours, plates were developed as previously
described.sup.22, dried overnight at RT, then sent to Zellnet
Consulting, New York, N.Y. for quantification. SFC and input cell
numbers were plotted, and a linear regression calculated after
excluding plateau data points.
[0111] Iii. TCR Avidity Assessment
[0112] TCR avidity was assessed by IFN ELIspot. 2.times.10.sup.5
CTLs were stimulated with serial dilutions of pepmixes (pp65,
Hexon) or 9mer peptides (NLV-pp65: NLVPMVATV HLA-A2 restricted,
QYD-pp65: QYDPVAALF HLA-A24 restricted; TDL-Hexon: TDLGQNLLY HLA-A1
restricted). The frequency of T cells specific for each
antigen/peptide was expressed as a percentage of the maximal
SFC/input cell number.
[0113] iv. Chromium Release Assay
[0114] The inventors measured the cytotoxic specificity in a
standard 4 hr Cr.sup.51 release assay, using E:T ratios of 40:1,
20:1, 10:1, and 5:1. CTLs were used as effectors and the targets
were PHA blasts pulsed with pepmixes. Autologous and allogeneic PHA
blasts alone or loaded with an irrelevant pepmix were used as
specificity and alloreactivity controls. The percentage of specific
lysis was calculated as [(experimental release-spontaneous
release)/(maximum release-spontaneous release)].times.100.
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incorporated by reference to the same extent as if each individual
publication was specifically and individually indicated to be
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[0167] Although the present invention and its advantages have been
described in detail, it should be understood that various changes,
substitutions and alterations can be made herein without departing
from the spirit and scope of the invention as defined by the
appended claims. Moreover, the scope of the present application is
not intended to be limited to the particular embodiments of the
process, machine, manufacture, composition of matter, means,
methods and steps described in the specification. As one of
ordinary skill in the art will readily appreciate from the
disclosure of the present invention, processes, machines,
manufacture, compositions of matter, means, methods, or steps,
presently existing or later to be developed that perform
substantially the same function or achieve substantially the same
result as the corresponding embodiments described herein may be
utilized according to the present invention. Accordingly, the
appended claims are intended to include within their scope such
processes, machines, manufacture, compositions of matter, means,
methods, or steps.
* * * * *